Methods and compositions for treating solid tumors and enhancing tumor vaccines

ABSTRACT

The present invention provides methods of treating and enhancing efficacy of immunotherapy for a solid tumor in a subject, comprising the step of contacting the subject with a compound or composition that modulates the expression or activity of ETRB, ET-1, ICAM-1, or another protein found herein to play a role in homing of T cells to a solid tumor. The present invention also provides methods of prognosticating a solid tumor in a subject, comprising the step of measuring an expression level of a protein found herein to play a role in homing of T cells to a solid tumor, or a nucleotide molecule encoding same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/001,241 (now U.S. Pat. No. 10,174,120) filed Jan. 20, 2016, which isa continuation of U.S. patent application Ser. No. 12/076,759 (now U.S.Pat. No. 9,289,426) filed Mar. 21, 2008, which claims priority from U.S.Provisional Patent Applications 60/907,091, filed Mar. 21, 2007 and60/907,138, filed Mar. 22, 2007, all of which are incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersCA098951, CA083638, HD043459, and TW000671 awarded by the NationalInstitutes of Health.

The government has certain rights in the invention.

FIELD OF INVENTION

The present invention provides methods of treating and enhancingefficacy of immunotherapy for a solid tumor in a subject, comprising thestep of contacting the subject with a compound or composition thatmodulates the expression or activity of ETRB, ET-1, ICAM-1, or anotherprotein found herein to play a role in homing of T cells to a solidtumor. The present invention also provides methods of prognosticating asolid tumor in a subject, comprising the step of measuring an expressionlevel of a protein found herein to play a role in homing of T cells to asolid tumor, or a nucleotide molecule encoding same.

BACKGROUND OF THE INVENTION

Clinical studies have demonstrated the potential of cancer immunetherapy using adoptively transferred T cells or tumor vaccines. Althoughthese have achieved marked response in some patients, they have fallenshort of expectations in others. The success of cell-mediated immunerejection mechanisms depends in part on the ability of effector cells toadequately infiltrate tumors. Yet, the mechanisms governing homing ofeffector cells into tumors remain poorly understood. Specifically, therole of endothelium in T cell homing to tumors has not been elucidatedto date.

Evidence exists that a variety of solid human tumors, includingmelanoma, gastrointestinal, breast, lung and ovarian cancer, arespontaneously infiltrated by T cells. Within each tumor type, theintensity of tumor-infiltrating T cells may vary significantly, andbrisk T cell infiltrate has been associated with improved prognosis. Forexample, T cells infiltrating tumor islets (intraepithelial T cells) aredetected only in a select group of patients in ovarian cancer. Thesepatients exhibit markedly improved progression-free and overallsurvival, a finding recently confirmed by others.

Methods for improving cancer vaccine immunotherapy are urgently neededin the art.

SUMMARY OF THE INVENTION

The present invention provides methods of treating and enhancingefficacy of immunotherapy for a solid tumor in a subject, comprising thestep of contacting the subject with a compound or composition thatmodulates the expression or activity of ETRB, ET-1, ICAM-1, or anotherprotein found herein to play a role in homing of T cells to a solidtumor. The present invention also provides methods of prognosticating asolid tumor in a subject, comprising the step of measuring an expressionlevel of a protein found herein to play a role in homing of T cells to asolid tumor, or a nucleotide molecule encoding same.

In one embodiment, provided herein is a method of treating a solid tumorin a subject, comprising the step of contacting the subject with acompound or composition that reduces an activity of an Endothelin Breceptor (ETRB), thereby treating a solid tumor in a subject. In anotherembodiment, the solid tumor is an ovarian tumor. In another embodiment,the solid tumor is an epithelial ovarian tumor. In another embodiment,the solid tumor is any other type of solid tumor known in the art. Inanother embodiment, the tumor is contacted by the compound orcomposition. Each possibility represents a separate embodiment of thepresent invention.

In one embodiment, provided herein is a method of enhancing an efficacyof an immunotherapy for a solid tumor in a subject, comprising the stepof contacting the subject with a compound or composition that reduces anactivity of an ETRB, thereby enhancing an efficacy of an immunotherapyfor a solid tumor in a subject. In another embodiment, the solid tumoris an ovarian tumor. In another embodiment, the solid tumor is anepithelial ovarian tumor. In another embodiment, the solid tumor is anyother type of solid tumor known in the art. In another embodiment, thetumor is contacted by the compound or composition. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that reduces an expression of an ETRB, therebytreating a solid tumor in a subject. In another embodiment, the solidtumor is an ovarian tumor. In another embodiment, the solid tumor is anepithelial ovarian tumor. In another embodiment, the solid tumor is anyother type of solid tumor known in the art. In another embodiment, thetumor is contacted by the compound or composition. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatreduces an expression of an ETRB, thereby enhancing an efficacy of animmunotherapy for a solid tumor in a subject. In another embodiment, thesolid tumor is an ovarian tumor. In another embodiment, the solid tumoris an epithelial ovarian tumor. In another embodiment, the solid tumoris any other type of solid tumor known in the art. In anotherembodiment, the tumor is contacted by the compound or composition. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that reduces an expression of a protein selectedfrom Musashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, andSGCB, thereby treating a solid tumor in a subject. In anotherembodiment, the solid tumor is an ovarian tumor. In another embodiment,the solid tumor is an epithelial ovarian tumor. In another embodiment,the solid tumor is any other type of solid tumor known in the art. Inanother embodiment, the tumor is contacted by the compound orcomposition. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatreduces an expression of a protein selected from Musashi 2, delta-like1, Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609, ACTR6, cloneLNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2,HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792,IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB, thereby enhancingan efficacy of an immunotherapy for a solid tumor in a subject. Inanother embodiment, the solid tumor is an ovarian tumor. In anotherembodiment, the solid tumor is an epithelial ovarian tumor. In anotherembodiment, the solid tumor is any other type of solid tumor known inthe art. In another embodiment, the tumor is contacted by the compoundor composition. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that reduces an activity of a protein selectedfrom Musashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, andSGCB, thereby treating a solid tumor in a subject. In anotherembodiment, the solid tumor is an ovarian tumor. In another embodiment,the solid tumor is an epithelial ovarian tumor. In another embodiment,the solid tumor is any other type of solid tumor known in the art. Inanother embodiment, the tumor is contacted by the compound orcomposition. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of comprising the step of contacting the subject with acompound or composition that reduces an activity of a protein selectedfrom Musashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, MPHOSPH9, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3,SLC1A4, and SGCB, thereby enhancing an efficacy of an immunotherapy fora solid tumor in a subject. In another embodiment, the solid tumor is anovarian tumor. In another embodiment, the solid tumor is an epithelialovarian tumor. In another embodiment, the solid tumor is any other typeof solid tumor known in the art. In another embodiment, the tumor iscontacted by the compound or composition. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that increases an expression of a proteinselected from CASP8 and FADD-like apoptosis regulator (CFLAR) protein;estrogen receptor alpha (ESR1); caldesmin-1; adrenergic receptor B2(ADRBK2); IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405,CYP1B1, EIFSB, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300, F1110330,C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10, therebytreating a solid tumor in a subject. In another embodiment, the solidtumor is an ovarian tumor. In another embodiment, the solid tumor is anepithelial ovarian tumor. In another embodiment, the solid tumor is anyother type of solid tumor known in the art. In another embodiment, thetumor is contacted by the compound or composition. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatincreases an expression of a protein selected from CFLAR; ESR1;caldesmin-1, ADRBK2; IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521,COL05405, CYP1B1, EIFSB, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300,F1110330, C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL,NOL7, MYCBP2, IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10,thereby enhancing an efficacy of an immunotherapy for a solid tumor in asubject. In another embodiment, the solid tumor is an ovarian tumor. Inanother embodiment, the solid tumor is an epithelial ovarian tumor. Inanother embodiment, the solid tumor is any other type of solid tumorknown in the art. In another embodiment, the tumor is contacted by thecompound or composition. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that increases an activity of a protein selectedfrom CFLAR; ESR1; caldesmin-1, ADRBK2; IMAGE:2755380, ZNFN1A5,LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1, EIF5B, IMAGE:1518332,HSPC056, FLJ32949, IMAGE:244300, F1110330, C18orf14, IMAGE:2115041,GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2, IMAGE:2275600, ADRBK2,EST366269, SCAP2, STK3, and AKAP10, thereby treating a solid tumor in asubject. In another embodiment, the solid tumor is an ovarian tumor. Inanother embodiment, the solid tumor is an epithelial ovarian tumor. Inanother embodiment, the solid tumor is any other type of solid tumorknown in the art. In another embodiment, the tumor is contacted by thecompound or composition. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatincreases an activity of a protein selected from CFLAR; ESR1;caldesmin-1, ADRBK2; IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521,COL05405, CYP1B1, EIF5B, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300,F1110330, C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL,NOL7, MYCBP2, IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10,thereby enhancing an efficacy of an immunotherapy for a solid tumor in asubject. In another embodiment, the solid tumor is a solid tumor. Inanother embodiment, the solid tumor is an epithelial ovarian tumor. Inanother embodiment, the solid tumor is any other type of solid tumorknown in the art. In another embodiment, the tumor is contacted by thecompound or composition. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatreduces an expression or activity of an endothelin-1 (ET-1) protein,thereby enhancing an efficacy of an immunotherapy for a solid tumor in asubject. In another embodiment, the solid tumor is an ovarian tumor. Inanother embodiment, the solid tumor is an epithelial ovarian tumor. Inanother embodiment, the solid tumor is any other type of solid tumorknown in the art. In another embodiment, the tumor is contacted by thecompound or composition. In another embodiment, a cancer cell of thetumor is contacted by the compound or composition. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatreduces an interaction between an ETRB and ET-1, thereby enhancing anefficacy of an immunotherapy for a solid tumor in a subject. In anotherembodiment, the solid tumor is an ovarian tumor. In another embodiment,the solid tumor is an epithelial ovarian tumor. In another embodiment,the solid tumor is any other type of solid tumor known in the art. Inanother embodiment, the tumor is contacted by the compound orcomposition. In another embodiment, a cancer cell of the tumor iscontacted by the compound or composition. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from a chemotherapy priorto a oncologic surgery, the method comprising the steps of (a) measuringan expression level of an Endothelin B receptor (ETRB) or a nucleotidemolecule encoding an Endothelin B receptor (ETRB) in the solid tumor;and (b) comparing the expression level to a reference standard, whereby,if the expression level is higher than the reference standard, then thesubject is likely to benefit from the chemotherapy prior to theoncologic surgery. In another embodiment, the chemotherapy is aneoadjuvant chemotherapy. In another embodiment, the oncologic surgeryis a debulking surgery. In another embodiment, the surgery is acytoreductive surgery. In another embodiment, the surgery is apalliative surgery. In another embodiment, the surgery is a supportivesurgery. Each possibility represents a separate embodiment of thepresent invention. In another embodiment, the solid tumor is an ovariantumor. In another embodiment, the solid tumor is an epithelial ovariantumor. In another embodiment, the solid tumor is any other type of solidtumor known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of prognosticating asolid tumor in a subject, the method comprising the steps of (a)measuring an expression level of an ETRB or a nucleotide moleculeencoding an ETRB in the solid tumor; and (b) comparing the expressionlevel to a reference standard, whereby, if the expression level ishigher than the reference standard, then the prognosis is less favorablethan a subject for whom the expression level is lower than or equal tothe reference standard. In another embodiment, the solid tumor is anovarian tumor. In another embodiment, the solid tumor is an epithelialovarian tumor. In another embodiment, the solid tumor is any other typeof solid tumor known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from an immunotherapy, themethod comprising the steps of (a) measuring an expression level of anETRB or a nucleotide molecule encoding an ETRB in the solid tumor; and(b) comparing the expression level to a reference standard, whereby, ifthe expression level is lower than the reference standard, then thesubject is likely to benefit from an immunotherapy. In anotherembodiment, the solid tumor is an ovarian tumor. In another embodiment,the solid tumor is an epithelial ovarian tumor. In another embodiment,the solid tumor is any other type of solid tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatincreases an expression or activity of an intercellular adhesionmolecule 1 (ICAM-1) protein, thereby enhancing an efficacy of animmunotherapy for a solid tumor in a subject. In another embodiment, thesolid tumor is an ovarian tumor. In another embodiment, the solid tumoris an epithelial ovarian tumor. In another embodiment, the solid tumoris any other type of solid tumor known in the art. In anotherembodiment, the tumor is contacted by the compound or composition. Eachpossibility represents a separate embodiment of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be better understood from a reading of the followingdetailed description taken in conjunction with the drawings in whichlike reference designators are used to designate like elements, and inwhich

FIGS. 1A-1E: FIG. 1A. Immuno-LCM steps. Left panel: Rapid IHC for CD31allows prompt identification of vasculature in ovarian cancer frozensections. Middle panel: Tissue section after LCM of CD31⁺ cells. Rightpanel: captured tumor vascular cells. FIG. 1B. RT-PCR analysis oflineage-specific markers in RNA from tumor vascular cells (TVC) isolatedwith immuno-LCM, whole tumor (Tum) or a no template control (NTC). C.Scatter plot and correlation value of amplified RNA from unstainedtissue (Pre IHC) versus RNA amplified after IHC optimized as in Table 1(Post IHC). FIG. 1D-A. Part 1. Heat map condition tree developed using ahierarchical clustering algorithm, excluding all genes where thedifference between the means of the tumor and normal vascular sampleswas less than its standard error. FIG. 1D-B, part 2: Red color shownalone. D, part 3: Green color shown alone. FIGS. 1E-A and 1E-B. ArchivedGene Expression Datasets. Data used for expression of the TVM in normaland tumor tissue samples; also available in the Gene Expression Omnibus(GEO; National Center for Biotechnology Information [NCBI]) with seriesnumbers GSE3526 and GSE2109, respectively. All CEL-files were similarlyprocessed using the Robust Multi-array Average (RMA) algorithm.

FIGS. 2A-2G. Quality of total RNA isolated from 8 μm frozen ovariancancer tissue sections through different methodologies and analyzed byAgilent Bioanalyzer (FIG. 2A-FIG. 2E), quantitative real-time PCR (FIG.2F) or Affymetrix U133A arrays (FIG. 2G). FIG. 2A. RNA distributionprofiles following fixation with different fixatives. (Lanes: 1,ethanol; 2, methanol; 3, acetone; 4, acetic acid+ethanol; 5,paraformaldehyde). FIG. 2B. RNA profiles after isolation withoutimmunostaining (Lane 1) or following IHC with or without RNAseinhibitor. (Lanes: 2, RNA Protector; 3, Placental RNAse inhibitor; 4,SuperRNASin; 5, RNA Protector+SuperRNASin; 6, no RNAse inhibitor. FIG.2C. RNA profiles after different immunostaining procedures. (Lanes: 1,IHC using DAB; 2, IHC using AEC; 3, immunofluorescence). FIG. 2D. Timecourse demonstrating RNA stability after IHC performed with procedureoptimized as in Table 1. (Lanes: 1, 0 min; 2, 30 min; 3, one hr; 4, twohrs; 5, three hrs). FIG. 2E. RNA profiles following different RNAisolation methods. (Lanes: 1, Arcturus kit; 2, Stratagene kit; 3,modified Trizol; 4, Zymo kit). FIG. 2F. qRT-PCR for β-actin transcriptswith RNA purified with the indicated RNA purification protocols. FIG.2G. Scatter plots and correlation values of amplified RNA (y-axis) tounamplified total tumor RNA (x-axis).

FIGS. 3A-3B. Vascular cells from ITC(+) and ITC(−) tumors clusterseparately. FIG. 3A. Condition tree and heat map based on vascular cellRNA expression from normal vasculature (Blue), ITC(−) tumor vascularcells (Yellow) and ITC(+) tumor vascular cells (Red). Samples wereclassified using a list of genes previously identified to classify tumorversus normal vascular cells and then sorted based on high expression inITC(+) versus ITC(−) vascular samples. FIG. 3B. List of genesdifferentially expressed.

FIGS. 4A-4B. qRT-PCR confirmation of differential mRNA expression. FIG.4A. qRT-PCR of whole tumor RNA for the indicated genes from 28 stage IIIepithelial ovarian cancers 16 ITC(+) and 12 ITC(−). FIG. 4B. qRT-PCR forthe indicated genes on FACS isolated tumor endothelial cells from 4ITC(+) and 3 ITC(−) tumors. ** indicates statistically significantdifference between samples with p<0.05.

FIGS. 5A-5C. Confirmation of Protein Expression. FIG. 5A.Immunohistochemistry for ET_(B)R, confirming protein expression in tumorvascular cells from epithelial ovarian cancer. FIG. 5B. Western blotanalysis demonstrating increase ETRB protein ITC(−) tumors as comparedto ITC(+) tumors (Left panels) and increased ETRB expression in ITC(+)poor prognosis tumors (overall survival<36 months) (right panels). FIG.5C. A bar graph showing Endothelin-1 (ET-1) mRNA expression in ovariancancer with or without TIL (n=16 each, mean±SD, p=0.26).

FIGS. 6A-6B. FIG. 6A. ETRB as a Biomarker for Poor Prognosis in OvarianCancer. Disease-free and overall survival curves from a panel of 61stage III epithelial ovarian cancer patients based upon ITC status (+)vs (−) and based upon high or low ETRB mRNA expression level asdetermined by qRTPCR. FIG. 6B. A graph showing the impact of treatmentwith BQ-788, starting at 2 or at 5 weeks, on tumor growth.

FIGS. 7A-7C. ETRB inhibition restricts tumor growth and increasesoverall survival in vaccinated animals. FIG. 7A and FIG. 7B. Tumorgrowth curves for ID8 tumors injected in the flank of animals treatedwith either no therapy (Vaccine −), anti-tumor vaccine and controlprotein therapy (Vaccine+), no vaccine and BQ788 therapy, or vaccine andBQ788 therapy. Arrows indicate time of BQ788 or control proteinadministration at either 2 weeks (FIG. 7A) or 5 weeks after tumorengraftment (FIG. 7B). FIG. 7C. Overall survival curves of animalsinjected with intraperitoneal ID8 cells, which received eitheranti-tumor vaccine+control protein therapy (Vaccine+) or vaccine+BQ788therapy. BQ788 therapy was initiated two weeks after injection ofintraperitoneal ID8 cells.

FIG. 8A-8F. ETRB inhibition leads to increased CD8⁺ T-cell infiltrationinto tumors. FIG. 8A. IHC demonstrating few intratumoral CD8 positivecells in vaccinated control animals (left panels) but significantnumbers of CD8⁺ T cells after early or delayed administration of BQ788(middle and left panels). FIG. 8B FACS analysis demonstrating increasednumbers of CD3⁺, CD8⁺ T cells in BQ788 treated animals as compared tocontrol animals. FIG. 8C. T cell proliferation assay in response to ID8pulsed dendritic cells from vaccinated-BQ788 treated animals orcontrols. FIG. 8D. Cytotoxic T lymphocyte assay demonstrating theability of CD8⁺ splenocytes from vaccinated control and BQ788 vaccinatedanimals to lyse ID8 cells. FIG. 8E. A graph showing flow cytometricquantification of total CD8⁺ tetramer+ cells in TC-1 tumors from a mousetreated with vaccine plus BQ-788 or vaccine alone. FIG. 8F. Showsascites development in vaccinated animals treated with BQ-788 andanimals treated with control peptide.

FIG. 9A-9C. FIG. 9A. Morphologic changes observed in BQ788-treated HUVECin the presence of Endothelin, as compared to Endothelin only orEndothelin plus BQ123 treated HUVEC.

FIG. 9B. qRTPCR demonstrating increased expression of ICAM1 anddecreased expression of VE-Cadherin in Endothelin+BQ788-treated HUVEC ascompared to Endothelin alone, Endothelin+BQ788, or BQ788 alone treatedHUVEC. FIG. 9C. Demonstration of an increased ability of T cells toadhere to Endothelin+BQ788-treated HUVEC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating and enhancingefficacy of immunotherapy for a solid tumor in a subject, comprising thestep of contacting the subject with a compound or composition thatmodulates the expression or activity of ETRB, ET-1, ICAM-1, or anotherprotein found herein to play a role in homing of T cells to a solidtumor. The present invention also provides methods of prognosticating asolid tumor in a subject, comprising the step of measuring an expressionlevel of a protein found herein to play a role in homing of T cells to asolid tumor, or a nucleotide molecule encoding same.

In one embodiment, provided herein is a method of treating a solid tumorin a subject, comprising the step of contacting the subject with acompound or composition that reduces an activity of an Endothelin Breceptor (ETRB), thereby treating a solid tumor in a subject. In anotherembodiment, the solid tumor is an ovarian tumor. In another embodiment,the solid tumor is an epithelial ovarian tumor. In another embodiment,the solid tumor is any other type of solid tumor known in the art. Inanother embodiment, the tumor is contacted by the compound orcomposition. Each possibility represents a separate embodiment of thepresent invention.

In one embodiment, provided herein is a method of enhancing an efficacyof an immunotherapy for a solid tumor in a subject, comprising the stepof contacting the subject with a compound or composition that reduces anactivity of an ETRB, thereby enhancing an efficacy of an immunotherapyfor a solid tumor in a subject. In another embodiment, the solid tumoris an ovarian tumor. In another embodiment, the solid tumor is anepithelial ovarian tumor. In another embodiment, the solid tumor is anyother type of solid tumor known in the art. In another embodiment, thetumor is contacted by the compound or composition. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that reduces an expression of an ETRB, therebytreating a solid tumor in a subject. In another embodiment, the solidtumor is an ovarian tumor. In another embodiment, the solid tumor is anepithelial ovarian tumor. In another embodiment, the solid tumor is anyother type of solid tumor known in the art. In another embodiment, thetumor is contacted by the compound or composition. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatreduces an expression of an ETRB, thereby enhancing an efficacy of animmunotherapy for a solid tumor in a subject. In another embodiment, thesolid tumor is an ovarian tumor. In another embodiment, the solid tumoris an epithelial ovarian tumor. In another embodiment, the solid tumoris any other type of solid tumor known in the art. In anotherembodiment, the tumor is contacted by the compound or composition. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that reduces an expression of a protein selectedfrom Musashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, MPHOSPH9, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3,SLC1A4, and SGCB, thereby treating a solid tumor in a subject. Inanother embodiment, the solid tumor is an ovarian tumor. In anotherembodiment, the solid tumor is an epithelial ovarian tumor. In anotherembodiment, the solid tumor is any other type of solid tumor known inthe art. In another embodiment, the tumor is contacted by the compoundor composition. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatreduces an expression of a protein selected from Musashi 2, delta-like1, Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609, ACTR6, cloneLNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2,HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792,IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB, thereby enhancingan efficacy of an immunotherapy for a solid tumor in a subject. Inanother embodiment, the solid tumor is an ovarian tumor. In anotherembodiment, the solid tumor is an epithelial ovarian tumor. In anotherembodiment, the solid tumor is any other type of solid tumor known inthe art. In another embodiment, the tumor is contacted by the compoundor composition. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that reduces an activity of a protein selectedfrom Musashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, MPHOSPH9, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3,SLC1A4, and SGCB, thereby treating a solid tumor in a subject. Inanother embodiment, the solid tumor is an ovarian tumor. In anotherembodiment, the solid tumor is an epithelial ovarian tumor. In anotherembodiment, the solid tumor is any other type of solid tumor known inthe art. In another embodiment, the tumor is contacted by the compoundor composition. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of comprising the step of contacting the subject with acompound or composition that reduces an activity of a protein selectedfrom Musashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, MPHOSPH9, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3,SLC1A4, and SGCB, thereby enhancing an efficacy of an immunotherapy fora solid tumor in a subject. In another embodiment, the solid tumor is anovarian tumor. In another embodiment, the solid tumor is an epithelialovarian tumor. In another embodiment, the solid tumor is any other typeof solid tumor known in the art. In another embodiment, the tumor iscontacted by the compound or composition. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that increases an expression of a proteinselected from CASP8 and FADD-like apoptosis regulator (CFLAR) protein;estrogen receptor alpha (ESR1); caldesmin-1; adrenergic receptor B2(ADRBK2); C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521,COL05405, CYP1B1, EIFSB, IMAGE:1518332, HSPC056, F1132949, IMAGE:244300,F1110330, C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL,NOL7, MYCBP2, IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10,thereby treating a solid tumor in a subject. In another embodiment, thesolid tumor is an ovarian tumor. In another embodiment, the solid tumoris an epithelial ovarian tumor. In another embodiment, the solid tumoris any other type of solid tumor known in the art. In anotherembodiment, the tumor is contacted by the compound or composition. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatincreases an expression of a protein selected from CFLAR; ESR1;caldesmin-1, ADRBK2; C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3,ZNF521, COL05405, CYP1B1, EIF5B, IMAGE:1518332, HSPC056, FLJ32949,IMAGE:244300, FLJ10330, C18orf14, IMAGE:2115041, GBP1, IMAGE:731714,SFRS1, NICAL, NOL7, MYCBP2, IMAGE:2275600, ADRBK2, EST366269, SCAP2,STK3, and AKAP10, thereby enhancing an efficacy of an immunotherapy fora solid tumor in a subject. In another embodiment, the solid tumor is anovarian tumor. In another embodiment, the solid tumor is an epithelialovarian tumor. In another embodiment, the solid tumor is any other typeof solid tumor known in the art. In another embodiment, the tumor iscontacted by the compound or composition. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, provided herein is a method of treating a solidtumor in a subject, comprising the step of contacting the subject with acompound or composition that increases an activity of a protein selectedfrom CFLAR; ESR1; caldesmin-1, ADRBK2; C3, IMAGE:2755380, ZNFN1A5,LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1, EIF5B, IMAGE:1518332,HSPC056, FLJ32949, IMAGE:244300, F1110330, C18orf14, IMAGE:2115041,GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2, IMAGE:2275600, ADRBK2,EST366269, SCAP2, STK3, and AKAP10, thereby treating a solid tumor in asubject. In another embodiment, the solid tumor is an ovarian tumor. Inanother embodiment, the solid tumor is an epithelial ovarian tumor. Inanother embodiment, the solid tumor is any other type of solid tumorknown in the art. In another embodiment, the tumor is contacted by thecompound or composition. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatincreases an activity of a protein whereby the compound is or thecomposition comprises CFLAR. In another embodiment, the compound is ESR1. . . . In another embodiment, the compound is caldesmin-1 . . . . Inanother embodiment, the compound is ADRBK2 . . . . In anotherembodiment, the compound is C3 . . . . In another embodiment, thecompound is IMAGE:2755380 . . . . In another embodiment, the compound isZNFN1A5 . . . . In another embodiment, the compound is LOC283663 . . . .In another embodiment, the compound is IGLJ3. ZNF521 . . . . In anotherembodiment, the compound is COL05405 . . . . In another embodiment, thecompound is CYP1B1 . . . . In another embodiment, the compound is EIF5B. . . . In another embodiment, the compound is IMAGE . . . . In anotherembodiment, the compound is 1518332 . . . . In another embodiment, thecompound is HSPC056 . . . . In another embodiment, the compound isFLJ32949 . . . . In another embodiment, the compound is IMAGE:244300 . .. . In another embodiment, the compound is FLJ10330 . . . . In anotherembodiment, the compound is C18orf14 . . . . In another embodiment, thecompound is IMAGE:2115041 . . . . In another embodiment, the compound isGBP1 . . . . In another embodiment, the compound is IMAGE:731714 . . . .In another embodiment, the compound is SFRS1 . . . . In anotherembodiment, the compound is NICAL . . . . In another embodiment, thecompound is NOL7 . . . . In another embodiment, the compound is MYCBP2 .. . . In another embodiment, the compound is IMAGE:2275600 . . . . Inanother embodiment, the compound is ADRBK2 . . . . In anotherembodiment, the compound is EST366269 . . . . In another embodiment, thecompound is SCAP2 . . . . In another embodiment, the compound is STK3 .. . . In another embodiment, the compound is AKAP10. Thereby enhancingan efficacy of an immunotherapy for a solid tumor in a subject. Inanother embodiment, the solid tumor is a solid tumor. In anotherembodiment, the solid tumor is an epithelial ovarian tumor. In anotherembodiment, the solid tumor is any other type of solid tumor known inthe art. In another embodiment, the tumor is contacted by the compoundor composition. Each possibility represents a separate embodiment of thepresent invention.

In one embodiment the compound used in the compositions described hereinfor increasing the activity of a protein selected is CFLAR, or ESR1;caldesmin-1, ADRBK2; C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3,ZNF521, COL05405, CYP1B1, EIFSB, IMAGE:1518332, HSPC056, FLJ32949,IMAGE:244300, F1110330, C18orf14, IMAGE:2115041, GBP1, IMAGE:731714,SFRS1, NICAL, NOL7, MYCBP2, IMAGE:2275600, ADRBK2, EST366269, SCAP2,STK3, AKAP10, or their combination in other discrete embodiments of thecompounds used in the methods and compositions provided herein.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatreduces an expression or activity of an endothelin-1 (ET-1) protein,thereby enhancing an efficacy of an immunotherapy for a solid tumor in asubject. In another embodiment, the solid tumor is an ovarian tumor. Inanother embodiment, the solid tumor is an epithelial ovarian tumor. Inanother embodiment, the solid tumor is any other type of solid tumorknown in the art. In another embodiment, the tumor is contacted by thecompound or composition. In another embodiment, a cancer cell of thetumor is contacted by the compound or composition. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatreduces an interaction between an ETRB and ET-1, thereby enhancing anefficacy of an immunotherapy for a solid tumor in a subject. In anotherembodiment, the solid tumor is an ovarian tumor. In another embodiment,the solid tumor is an epithelial ovarian tumor. In another embodiment,the solid tumor is any other type of solid tumor known in the art. Inanother embodiment, the tumor is contacted by the compound orcomposition. In another embodiment, a cancer cell of the tumor iscontacted by the compound or composition. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from a chemotherapy priorto a oncologic surgery, the method comprising the steps of (a) measuringan expression level of an Endothelin B receptor (ETRB) or a nucleotidemolecule encoding an Endothelin B receptor (ETRB) in the solid tumor;and (b) comparing the expression level to a reference standard, whereby,if the expression level is higher than the reference standard, then thesubject is likely to benefit from the chemotherapy prior to theoncologic surgery. In another embodiment, the ETRB level is measured ina tumor endothelial cell (TEC) or TEC population of the tumor. Inanother embodiment, the chemotherapy is a neoadjuvant chemotherapy. Inanother embodiment, the oncologic surgery is a debulking surgery. Inanother embodiment, the surgery is a cytoreductive surgery. In anotherembodiment, the surgery is a palliative surgery. In another embodiment,the surgery is a supportive surgery. In another embodiment, the solidtumor is an ovarian tumor. In another embodiment, the solid tumor is anepithelial ovarian tumor. In another embodiment, the solid tumor is anyother type of solid tumor known in the art. Each possibility representsa separate embodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from a chemotherapy priorto a oncologic surgery, the method comprising the steps of (a) measuringan expression level of an ET-1 or a nucleotide molecule encoding an ET-1in the solid tumor; and (b) comparing the expression level to areference standard, whereby, if the expression level is higher than thereference standard, then the subject is likely to benefit from thechemotherapy prior to the oncologic surgery. In another embodiment, thesolid tumor is an ovarian tumor. In another embodiment, the solid tumoris an epithelial ovarian tumor. In another embodiment, the solid tumoris any other type of solid tumor known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from a chemotherapy priorto a oncologic surgery, the method comprising the steps of (a) measuringan expression level in the solid tumor of a protein selected fromMusashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, MPHOSPH9, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3,SLC1A4, and SGCB; or a nucleotide molecule encoding a protein selectedfrom Musashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, MPHOSPH9, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3,SLC1A4, and SGCB; and (b) comparing the expression level to a referencestandard, whereby, if the expression level is higher than the referencestandard, then the subject is likely to benefit from the chemotherapyprior to the oncologic surgery. In another embodiment, the expressionlevel is measured in a TEC or TEC population of the tumor. In anotherembodiment, the solid tumor is an ovarian tumor. In another embodiment,the solid tumor is an epithelial ovarian tumor. In another embodiment,the solid tumor is any other type of solid tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from a chemotherapy priorto a oncologic surgery, the method comprising the steps of (a) measuringan expression level in the solid tumor of a protein selected from CFLAR;ESR1; caldesmin-1, ADRBK2; C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3,ZNF521, COL05405, CYP1B1, EIF5B, IMAGE:1518332, HSPC056, FLJ32949,IMAGE:244300, FLJ10330, C18orf14, IMAGE:2115041, GBP1, IMAGE:731714,SFRS1, NICAL, NOL7, MYCBP2, IMAGE:2275600, ADRBK2, EST366269, SCAP2,STK3, and AKAP10 or a nucleotide molecule encoding a protein selectedfrom CFLAR; ESR1; caldesmin-1, ADRBK2; C3, IMAGE:2755380, ZNFN1A5,LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1, EIFSB, IMAGE:1518332,HSPC056, FLJ32949, IMAGE:244300, F1110330, C18orf14, IMAGE:2115041,GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2, IMAGE:2275600, ADRBK2,EST366269, SCAP2, STK3, and AKAP10; and (b) comparing the expressionlevel to a reference standard, whereby, if the expression level is lowerthan the reference standard, then the subject is likely to benefit fromthe chemotherapy prior to the oncologic surgery. In another embodiment,the expression level is measured in a TEC or TEC population of thetumor. In another embodiment, the solid tumor is an ovarian tumor. Inanother embodiment, the solid tumor is an epithelial ovarian tumor. Inanother embodiment, the solid tumor is any other type of solid tumorknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, “chemotherapy” refers to neoadjuvantchemotherapy. In another embodiment, the term refers to any other typeof chemotherapy known in the art. In another embodiment, “oncologicsurgery” refers to a debulking surgery. In another embodiment, thesurgery is a cytoreductive surgery. In another embodiment, the surgeryis a palliative surgery. In another embodiment, the surgery is asupportive surgery. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, provided herein is a method of prognosticating asolid tumor in a subject, the method comprising the steps of (a)measuring an expression level of an ETRB or a nucleotide moleculeencoding an ETRB in the solid tumor; and (b) comparing the expressionlevel to a reference standard, whereby, if the expression level ishigher than the reference standard, then the prognosis is less favorablethan a subject for whom the expression level is lower than or equal tothe reference standard. In another embodiment, the ETRB level ismeasured in a TEC or TEC population of the tumor. In another embodiment,the solid tumor is an ovarian tumor. In another embodiment, the solidtumor is an epithelial ovarian tumor. In another embodiment, the solidtumor is any other type of solid tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of prognosticating asolid tumor in a subject, the method comprising the steps of (a)measuring an expression level of an ET-1 or a nucleotide moleculeencoding an ET-1 in the solid tumor; and (b) comparing the expressionlevel to a reference standard, whereby, if the expression level ishigher than the reference standard, then the prognosis is less favorablethan a subject for whom the expression level is lower than or equal tothe reference standard. In another embodiment, the solid tumor is anovarian tumor. In another embodiment, the solid tumor is an epithelialovarian tumor. In another embodiment, the solid tumor is any other typeof solid tumor known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of prognosticating asolid tumor in a subject, the method comprising the steps of (a)measuring an expression level in the solid tumor of a protein selectedfrom Musashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, MPHOSPH9, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3,SLC1A4, and SGCB; or a nucleotide molecule encoding a protein selectedfrom Musashi 2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G,KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1,PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2,MYBL1, MPHOSPH9, IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3,SLC1A4, and SGCB; and (b) comparing the expression level to a referencestandard, whereby, if the expression level is higher than the referencestandard, then the prognosis is less favorable than a subject for whomthe expression level is lower than or equal to the reference standard.In another embodiment, the expression level is measured in a TEC or TECpopulation of the tumor. In another embodiment, the solid tumor is anovarian tumor. In another embodiment, the solid tumor is an epithelialovarian tumor. In another embodiment, the solid tumor is any other typeof solid tumor known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of providing aprognosis on treatment of a subject having a solid tumor, the methodcomprising the steps of (a) measuring an expression level in the solidtumor of a protein selected from CFLAR; ESR1; caldesmin-1, ADRBK2; C3,IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1,EIF5B, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300, F1110330,C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10; or anucleotide molecule encoding a protein selected from CFLAR; ESR1;caldesmin-1, ADRBK2; C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3,ZNF521, COL05405, CYP1B1, EIF5B, IMAGE:1518332, HSPC056, FLJ32949,IMAGE:244300, FLJ10330, C18orf14, IMAGE:2115041, GBP1, IMAGE:731714,SFRS1, NICAL, NOL7, MYCBP2, IMAGE:2275600, ADRBK2, EST366269, SCAP2,STK3, and AKAP10; and (b) comparing the expression level to a referencestandard, whereby, if the expression level is higher than the referencestandard, then the prognosis is more favorable than a subject for whomthe expression level is lower than or equal to the reference standard.In another embodiment, the expression level is measured in a TEC or TECpopulation of the tumor. In another embodiment, the solid tumor is anovarian tumor. In another embodiment, the solid tumor is an epithelialovarian tumor. In another embodiment, the solid tumor is any other typeof solid tumor known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from an immunotherapy, themethod comprising the steps of (a) measuring an expression level of anETRB or a nucleotide molecule encoding an ETRB in the solid tumor; and(b) comparing the expression level to a reference standard, whereby, ifthe expression level is lower than the reference standard, then thesubject is likely to benefit from an immunotherapy. In anotherembodiment, the method identifies a subject likely to benefit fromimmunotherapy in the absence of BQ788. In another embodiment, a subjectexhibiting a high ETRB expression level is a candidate for immunotherapyin conjunction with BQ788. In another embodiment, the expression levelis measured in a TEC or TEC population of the tumor. In anotherembodiment, the solid tumor is an ovarian tumor. In another embodiment,the solid tumor is an epithelial ovarian tumor. In another embodiment,the solid tumor is any other type of solid tumor known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from an immunotherapy, themethod comprising the steps of (a) measuring an expression level of anET-1 or a nucleotide molecule encoding an ET-1 in the solid tumor; and(b) comparing the expression level to a reference standard, whereby, ifthe expression level is lower than the reference standard, then thesubject is likely to benefit from an immunotherapy. In anotherembodiment, the method identifies a subject likely to benefit fromimmunotherapy in the absence of BQ788. In another embodiment, a subjectexhibiting a high ET-1 expression level is a candidate for immunotherapyin conjunction with BQ788. In another embodiment, the solid tumor is anovarian tumor. In another embodiment, the solid tumor is an epithelialovarian tumor. In another embodiment, the solid tumor is any other typeof solid tumor known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from an immunotherapy, themethod comprising the steps of (a) measuring an expression level in thesolid tumor of a protein selected from Musashi 2, delta-like 1,Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609, ACTR6, cloneLNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2,HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792,IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB; or a nucleotidemolecule encoding a protein selected from Musashi 2, delta-like 1,Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609, ACTR6, cloneLNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2,HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792,IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB; and (b) comparingthe expression level to a reference standard, whereby, if the expressionlevel is lower than the reference standard, then the subject is likelyto benefit from an immunotherapy. In another embodiment, the methodidentifies a subject likely to benefit from immunotherapy in the absenceof BQ788. In another embodiment, a subject exhibiting a high expressionlevel is a candidate for immunotherapy in conjunction with BQ788. Inanother embodiment, the expression level is measured in a TEC or TECpopulation of the tumor. In another embodiment, the solid tumor is anovarian tumor. In another embodiment, the solid tumor is an epithelialovarian tumor. In another embodiment, the solid tumor is any other typeof solid tumor known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, provided herein is a method of identifying asubject with a solid tumor likely to benefit from an immunotherapy, themethod comprising the steps of (a) measuring an expression level in thesolid tumor of a protein selected from CFLAR; ESR1; caldesmin-1, ADRBK2;C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1,EIF5B, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300, F1110330,C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10; or anucleotide molecule encoding a protein selected from CFLAR; ESR1;caldesmin-1, ADRBK2; C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3,ZNF521, COL05405, CYP1B1, EIF5B, IMAGE:1518332, HSPC056, FLJ32949,IMAGE:244300, FLJ10330, C18orf14, IMAGE:2115041, GBP1, IMAGE:731714,SFRS1, NICAL, NOL7, MYCBP2, IMAGE:2275600, ADRBK2, EST366269, SCAP2,STK3, and AKAP10; and (b) comparing the expression level to a referencestandard, whereby, if the expression level is lower than the referencestandard, then the subject is likely to benefit from an immunotherapy.In another embodiment, the method identifies a subject likely to benefitfrom immunotherapy in the absence of BQ788. In another embodiment, asubject exhibiting a high expression level is a candidate forimmunotherapy in conjunction with BQ788. In another embodiment, theexpression level is measured in a TEC or TEC population of the tumor. Inanother embodiment, the solid tumor is an ovarian tumor. In anotherembodiment, the solid tumor is an epithelial ovarian tumor. In anotherembodiment, the solid tumor is any other type of solid tumor known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatincreases an expression of an intercellular adhesion molecule 1 (ICAM-1)protein, thereby enhancing an efficacy of an immunotherapy for a solidtumor in a subject. In another embodiment, the tumor is contacted withthe compound or composition. In another embodiment, the solid tumor isan ovarian tumor. In another embodiment, the solid tumor is anepithelial ovarian tumor. In another embodiment, the solid tumor is anyother type of solid tumor known in the art. In another embodiment, thetumor is contacted by the compound or composition. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting the subject with a compound or composition thatincreases an activity of an ICAM-1 protein, thereby enhancing anefficacy of an immunotherapy for a solid tumor in a subject. In anotherembodiment, the tumor is contacted with the compound or composition. Inanother embodiment, the solid tumor is an ovarian tumor. In anotherembodiment, the solid tumor is an epithelial ovarian tumor. In anotherembodiment, the solid tumor is any other type of solid tumor known inthe art. In another embodiment, the tumor is contacted by the compoundor composition. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, provided herein is a method of inhibiting tumorgrowth in a subject, comprising the step of administering to the subjecta compound or composition that decreases the expression or activity of aprotein selected from ETRB, ET-1, Musashi 2, delta-like 1,Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609, ACTR6, cloneLNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2,HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792,IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB, thereby inhibitingtumor growth in a subject.

In another embodiment, provided herein is a method of inhibiting tumorgrowth in a subject, comprising the step of administering to the subjecta compound or composition that increases the expression or activity of aprotein selected from CFLAR; ESR1; caldesmin-1, ADRBK2; C3,IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1,EIF5B, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300, F1110330,C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10, therebyinhibiting tumor growth in a subject.

In another embodiment, a method of the present invention is performedfollowing oncologic surgery. In another embodiment, the method isperformed following debulking surgery. In another embodiment, the methodis performed following administration of chemotherapy. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is a method of inhibiting growthof metastases in a subject, comprising the step of administering to thesubject a compound or composition that decreases the expression oractivity of a protein selected from ETRB, ET-1, Musashi 2, delta-like 1,Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609, ACTR6, cloneLNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2,HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792,IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB, thereby inhibitinggrowth of metastases in a subject.

In another embodiment, provided herein is a method of inhibiting growthof metastases in a subject, comprising the step of administering to thesubject a compound or composition that increases the expression oractivity of a protein selected from CFLAR; ESR1; caldesmin-1, ADRBK2;C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1,EIFSB, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300, F1110330,C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10, therebyinhibiting growth of metastases in a subject.

In another embodiment, provided herein is a method of abrogatingtolerance of a subject to a tumor, comprising the step of administeringto the subject a compound or composition that decreases the expressionor activity of a protein selected from ETRB, ET-1, Musashi 2, delta-like1, Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609, ACTR6, cloneLNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2,HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792,IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB, thereby abrogatingtolerance of a subject to a tumor.

In another embodiment, provided herein is a method of abrogatingtolerance of a subject to a tumor, comprising the step of administeringto the subject a compound or composition that increases the expressionor activity of a protein selected from CFLAR; ESR1; caldesmin-1, ADRBK2;C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1,EIFSB, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300, F1110330,C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10, therebyabrogating tolerance of a subject to a tumor.

In another embodiment, provided herein is a method of increasing T cellhoming to a tumor, comprising the step of administering to the subject acompound or composition that decreases the expression or activity of aprotein selected from ETRB, ET-1, Musashi 2, delta-like 1,Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609, ACTR6, cloneLNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2,HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792,IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB, thereby increasingT cell homing to a tumor.

In another embodiment, provided herein is a method of increasing T cellhoming to a tumor, comprising the step of administering to the subject acompound or composition that increases the expression or activity of aprotein selected from CFLAR; ESR1; caldesmin-1, ADRBK2; C3,IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1,EIFSB, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300, F1110330,C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10, therebyincreasing T cell homing to a tumor.

In another embodiment, provided herein is a method of increasing T cellretention in a tumor islet, comprising the step of administering to thesubject a compound or composition that decreases the expression oractivity of a protein selected from ETRB, ET-1, Musashi 2, delta-like 1,Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609, ACTR6, cloneLNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2,HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792,IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB, thereby increasingT cell retention in a tumor islet.

In another embodiment, provided herein is a method of increasing T cellretention in a tumor islet, comprising the step of administering to thesubject a compound or composition that increases the expression oractivity of a protein selected from CFLAR; ESR1; caldesmin-1, ADRBK2;C3, IMAGE:2755380, ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1,EIFSB, IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300, F1110330,C18orf14, IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10, therebyincreasing T cell retention in a tumor islet.

In another embodiment, provided herein is an isolated CD8⁺ cell or cellpopulation isolated from a vaccinated BQ-788-treated animal. In anotherembodiment, the CD8⁺ cell or cell population is isolated from a tumor ofa vaccinated BQ-788-treated animal. In another embodiment, providedherein is a method of isolating a tumor-antigen specific T cell,comprising the step of administering an Endothelin antagonist to atumor-bearing animal. In another embodiment, the Endothelin antagonistis an ETRB antagonist. In another embodiment, the Endothelin antagonistis BQ-788. In another embodiment, the Endothelin antagonist is any othertype of Endothelin antagonist known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is an isolated CD8⁺ cell or cellpopulation isolated from an animal that has been treated with a compoundor composition the decreases the expression or activity of a proteinselected from ET-1, Musashi 2, delta-like 1, Hairy/Enhancer of Split 1,MEG3, SEC61G, KIAA1609, ACTR6, clone LNG00414, ATP9A, IMAGE:23539,NCOA1, WIT1, PAPSS2, ALDOA, ZNF423, ENPP2, HSU79266, KIAA0146,IMAGE:1902075, EMX2, MYBL1, MPHOSPH9, IMAGE:1660792, IMAGE:191524,IMAGE:2365035, TAF3, SLC1A4, and SGCB. In another embodiment, the CD8⁺cell or cell population is isolated from a tumor of the animal. Inanother embodiment, provided herein is a method of isolating atumor-antigen specific T cell, comprising the step of administering thecompound or composition to a tumor-bearing animal. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, provided herein is an isolated CD8⁺ cell or cellpopulation isolated from an animal that has been treated with a compoundor composition the increases the expression or activity of a proteinselected from CFLAR; ESR1; caldesmin-1, ADRBK2; C3, IMAGE:2755380,ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1, EIF5B,IMAGE:1518332, HSPC056, F1132949, IMAGE:244300, F1110330, C18orf14,IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10, therebyincreasing T cell retention in a tumor islet. In another embodiment, theCD8⁺ cell or cell population is isolated from a tumor of the animal. Inanother embodiment, provided herein is a method of isolating atumor-antigen specific T cell, comprising the step of administering thecompound or composition to a tumor-bearing animal. Each possibilityrepresents a separate embodiment of the present invention.

The animal used in methods and compositions of the present invention is,in another embodiment, a mouse. In another embodiment, the animal is arodent. In another embodiment, the animal is any animal used forresearch purposes. In another embodiment, the animal is any othersuitable animal known in the art.

In another embodiment, provided herein is a method of enhancing theeffectiveness of a tumor immunotherapy in a subject, comprising the stepof administering to the subject a composition that reduces theexpression or activity of RGC32, thereby enhancing the effectiveness ofa tumor immunotherapy in a subject.

In another embodiment, provided herein is a method of enhancing theeffectiveness of a tumor immunotherapy in a subject, comprising the stepof administering to the subject a composition that reduces theexpression or activity of VE-Cadherin, thereby enhancing theeffectiveness of a tumor immunotherapy in a subject.

In another embodiment, provided herein is a method of enhancing theeffectiveness of a tumor immunotherapy in a subject, comprising the stepof inhibiting an ETRB-mediated pathway, thereby enhancing theeffectiveness of a tumor immunotherapy in a subject. In anotherembodiment, the pathway is an intracellular pathway. In anotherembodiment, the pathway is an extracellular pathway. In anotherembodiment, the production of nitric oxide (NO) is inhibited. In anotherembodiment, the production of extracellular Ca2⁺ is inhibited. Inanother embodiment, the production of prostacyclin is inhibited. Inanother embodiment, the production of endothelium-derivedhyperpolarizing factor is inhibited. In another embodiment, the ETRBpathway that is inhibited is a G-protein-coupled receptor (GPCR)pathway. In another embodiment, the pathway involves activation ofphospholipase C by the GPCR. In another embodiment, the pathway involvesgeneration of inositol triphosphate from the phospholipase C. In anotherembodiment, the pathway involves generation of diacylglycerol from thephospholipase C. In another embodiment, the inositol triphosphatestimulates calcium release. In another embodiment, the diacylglycerolcauses protein kinase C activation. In another embodiment, the ETRBpathway that is a phospholipase D pathway. In another embodiment,diacylglycerol is generated by the phospholipase D activation. Inanother embodiment, phospholipase A2 is stimulated by the phospholipaseD activation. In another embodiment, arachidonic acid is releasedfollowing phospholipase A2 stimulation. In another embodiment, theNa+/H+ exchanger is activated by the phospholipase D. In anotherembodiment, a tyrosine kinase is activated by the phospholipase D. Inanother embodiment, a MAP kinase is activated by the phospholipase D.Each possibility represents a separate embodiment of the presentinvention.

Endothelin receptor-activated pathways are well known in the art, andare described for example, in Ignarro et al (Ignarro L J, Buga G M, WoodK S, Byrns R E, Chaudhuri G. Proc Natl Acad Sci USA. 1987;84:9265-9269); Furchgott et al (Furchgott R F, Vanhoutte P M. FASEB J.1989; 3:2007-2018); Fleming et al (Fleming I, Busse R. J Mol CellCardiol. 1999; 31:5-14); Vanhoutte et al (Vanhoutte P M. Nature. 1998;396:213, 215-216); and Brandes et al (Brandes R P, Schmitz-Winnenthal FH, Feletou M, Godecke A, Huang P L, Vanhoutte P M, Fleming I, Busse R.Proc Natl Acad Sci USA. 2000; 97:9747-9752). Each possibility representsa separate embodiment of the present invention.

In another embodiment of methods of the present invention, the compoundor composition is brought into contact with the solid tumor. In anotherembodiment, a tumor endothelial cells (TEC) of the solid tumor iscontacted. In another embodiment, an endothelial cell of the solid tumorcontacted. In another embodiment, wherein an ovarian tumor is thetarget, the ovarian tumor is contacted. In another embodiment, a TEC ofthe ovarian tumor is contacted. In another embodiment, an endothelialcell of the ovarian tumor contacted. In another embodiment, the compoundor composition is administered systemically. In another embodiment, thecompound or composition is administered directly to the tumor. Inanother embodiment, the compound or composition is administered in thevicinity of the tumor. Each possibility represents a separate embodimentof the present invention.

In another embodiment of methods of the present invention, the subjecthas received an immunotherapy. In another embodiment, the subject iscurrently receiving an immunotherapy. In another embodiment, the subjectis slated to receive an immunotherapy. “Currently receiving” refers, inanother embodiment, to a subject between doses of an immunotherapyregimen. In another embodiment, the term refers to a subject that hasreceived or will receive a dose of the immunotherapy regimen on the sameday as the method of the present invention is performed. In anotherembodiment, the subject receives a dose of the immunotherapy regimen inthe same week as the method of the present invention is performed. Inanother embodiment, the subject receives a dose of the immunotherapyregimen simultaneously with performing a method of the presentinvention. Each possibility represents a separate embodiment of thepresent invention.

“Immunotherapy” refers, in another embodiment, to a vaccine therapy. Inanother embodiment, the term refers to direct vaccination of thesubject. In another embodiment, the term refers to passive vaccinationof the subject. In another embodiment, the term refers to transfer tothe subject of a population of cells comprising anti-tumorantigen-specific T cells. In another embodiment, the population of cellsis from a donor. In another embodiment, the population of cells is fromthe subject. In another embodiment, the population of cells is expandedex vivo. In another embodiment, the anti-tumor antigen-specific T cellsin the population of cells are expanded ex vivo.

In another embodiment, the term refers to cytokine treatment. In anotherembodiment, the term refers to interferon treatment. In anotherembodiment, the term refers to growth factor treatment. In anotherembodiment, the term refers to antibody therapy. In another embodiment,the term refers to therapy with a compound that modulates T cellactivity. In another embodiment, the term refers to therapy with anadjuvant. In another embodiment, the term refers to adoptive lymphocytetherapy. In another embodiment, the term refers to cellularimmunotherapy. In another embodiment, the term refers to toll-likereceptor therapy. In another embodiment, the term refers to anytherapeutic method that utilizes an immune mechanism.

Each type of immunotherapy represents a separate embodiment of thepresent invention.

Methods for ex vivo immunotherapy are well known in the art and aredescribed, for example, in Davis I D et al (Blood dendritic cellsgenerated with Flt3 ligand and CD40 ligand prime CD8+ T cellsefficiently in cancer patients. J Immunother. 2006 September-October;29(5):499-511) and Mitchell M S et al (The cytotoxic T cell response topeptide analogs of the HLA-A*0201-restricted MUC1 signal sequenceepitope, M1.2. Cancer Immunol Immunother. 2006 Jul. 28). Each methodrepresents a separate embodiment of the present invention.

In another embodiment, “immunotherapy” comprises the steps of (a)inducing ex vivo, from human blood cells obtained from a donor,formation and proliferation of human CTL that recognize a malignant cellof the cancer; and (b) infusing the human CTL into the subject.

The anti-ETRB compound of methods and compositions of the presentinvention is, in another embodiment, BQ788. In another embodiment, thecompound is Bosentan (Tracleer™). In another embodiment, the compound istezosentan. In another embodiment, the compound is Pergolide. In anotherembodiment, the compound is any other anti-ETRB compound known in theart. In another embodiment, the compound is a general inhibitor ofEndothelin receptors. In another embodiment, the compound is specificfor ETRB. In another embodiment, the compound preferentially inhibitsETRB over other Endothelin receptors. In another embodiment, thecompound is an antibody. In another embodiment, the compound is ananti-ETRB antibody.

In another embodiment, the dose of BQ788 is below that used to inhibitangiogenesis.

Various embodiments of dosage ranges of BQ788 can be used, in anotherembodiment, in methods of the present invention. In one embodiment, thedosage is in the range of 1-80 mg/day. In another embodiment, the dosageis in the range of 5-80 mg/day. In another embodiment the dosage is inthe range of 20-80 mg/day. In another embodiment the dosage is in therange of 20-60 mg/day. In another embodiment the dosage is in the rangeof 40-60 mg/day. In another embodiment the dosage is in a range of 45-60mg/day. In another embodiment the dosage is in the range of 15-25mg/day. In another embodiment the dosage is in the range of 55-65mg/day. In one embodiment, the dosage is 20 mg/day. In anotherembodiment, the dosage is 40 mg/day. In another embodiment, the dosageis 60 mg/day. In another embodiment, the dosage is 80 mg/day.

In another embodiment, the dosage is 20 μg. In another embodiment, thedosage is 10 μg. In another embodiment, the dosage is 30 μg. In anotherembodiment, the dosage is 40 μg. In another embodiment, the dosage is 60μg. In another embodiment, the dosage is 80 μg. In another embodiment,the dosage is 100 μg. In another embodiment, the dosage is 150 μg. Inanother embodiment, the dosage is 200 μg. In another embodiment, thedosage is 300 μg. In another embodiment, the dosage is 400 μg. Inanother embodiment, the dosage is 600 μg. In another embodiment, thedosage is 800 μg. In another embodiment, the dosage is 1000 μg. Inanother embodiment, the dosage is 1500 μg. In another embodiment, thedosage is 2000 μg.

In another embodiment, the dosage is 10 μg/BQ788/dose. In anotherembodiment, the dosage is 20 μg/BQ788/dose. In another embodiment, thedosage is 30 μg/BQ788/dose. In another embodiment, the dosage is 40μg/BQ788/dose. In another embodiment, the dosage is 60 μg/BQ788/dose. Inanother embodiment, the dosage is 80 μg/BQ788/dose. In anotherembodiment, the dosage is 100 μg/BQ788/dose. In another embodiment, thedosage is 150 μg/BQ788/dose. In another embodiment, the dosage is 200μg/BQ788/dose. In another embodiment, the dosage is 300 μg/BQ788/dose.In another embodiment, the dosage is 400 μg/BQ788/dose. In anotherembodiment, the dosage is 600 μg/BQ788/dose. In another embodiment, thedosage is 800 μg/BQ788/dose. In another embodiment, the dosage is 1000μg/BQ788/dose. In another embodiment, the dosage is 1500 μg/BQ788/dose.In another embodiment, the dosage is 2000 μg/BQ788/dose.

In another embodiment, the BQ788 is administered systemically at 1 ofthe above doses. In another embodiment, the BQ788 is administeredintra-tumorally at 1 of the above doses. Each possibility represents aseparate embodiment of the present invention.

In another embodiment of methods and compositions of the presentinvention, the compound used to reduce expression of a protein is anantisense molecule. In another embodiment, the compound is an RNAinhibitory molecule. In another embodiment, the compound is any othertype of compound known in the art that is capable of reducing expressionof a protein or its transcript. Each possibility represents a separateembodiment of the present invention.

The step of “decreasing” the expression of a protein in a method of thepresent invention comprises, in another embodiment, directly decreasingthe protein level. In another embodiment, the step comprises inhibitingtranscription of the nucleotide molecule (e.g. mRNA) encoding theprotein. In another embodiment, the step comprises inhibitingtranslation of the mRNA. In another embodiment, the step comprisesinducing, enhancing, or increasing degradation of the mRNA. In anotherembodiment, the step comprises inducing, enhancing, or increasingdegradation of the protein itself. In another embodiment, the stepcomprises any other method of decreasing the expression of a gene orprotein that is known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a method of the present invention comprises theuse of a bivalent antibody that binds to both a therapeutic compound anda protein identified in the present invention. In another embodiment,the polyvalent antibody is conjugated to both a tumoricidal compound anda protein identified in the present invention. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, an anti-cancer agent is conjugated to a ligandthat binds a protein identified in the present invention or a nucleotideencoding same and administered to the subject. In another embodiment,the ligand is an antibody. In another embodiment, the ligand is acomplementary nucleotide molecule. In another embodiment, the ligand isa small molecule. In another embodiment, the ligand is any other type ofmolecule known in the art capable of binding a protein identified in thepresent invention or a nucleotide encoding same. Each possibilityrepresents a separate embodiment of the present invention.

The anti-cancer agent utilized in methods and compositions of thepresent invention is, in another embodiment, a radioactive isotope. Inanother embodiment, the anti-cancer agent is a cytotoxic agent. Inanother embodiment, the anti-cancer agent is a cytotoxic drug. Inanother embodiment, the anti-cancer agent is a nucleic acid molecule. Inanother embodiment, the anti-cancer agent is an antisense molecule. Inanother embodiment, the anti-cancer agent is an RNA inhibitory molecule.In another embodiment, the anti-cancer agent is an anti-tumor agent. Inanother embodiment, the anti-cancer agent is a cytotoxic virus. Inanother embodiment, the anti-cancer agent is a cytotoxic pathogen. Inanother embodiment, the anti-cancer agent is a nanosphere. In anotherembodiment, the nanosphere is loaded with a cytotoxic compound. Inanother embodiment, the nanosphere is loaded with a chemotherapy drug.In another embodiment, the nanosphere is loaded with a toxin. In anotherembodiment, the nanosphere is loaded with an anti-cancer compound. Inanother embodiment, the anti-cancer agent is a nanoparticle. In anotherembodiment, the anti-cancer agent is an engineered T cell. In anotherembodiment, the anti-cancer agent is an engineered cytotoxic cell. Inanother embodiment, the anti-cancer agent is any other type ofengineered molecule known in the art. In another embodiment, theanti-cancer agent is any other agent used in cancer therapy. In anotherembodiment, the anti-cancer agent is any other type of anti-cancer agentknown in the art. Each possibility represents a separate embodiment ofthe present invention.

In one embodiment, virions whose tail tube major subunit (V) proteinsare modified with a cyclizable Arg-Gly-Asp (RGD) peptide are able totransfect tumor cells at a significant frequency. Phage-mediatedtransfection with virions whose tail tube major subunit (V) proteins aremodified with a cyclizable Arg-Gly-Asp (RGD) capable of expressing thecompounds described herein are used in one embodiment with thecompositions described herein for the treatment methods provided.

“Engineered T cell” refers, in another embodiment, to a T cell designedto recognize a cell containing or expressing a molecule of interest. Inanother embodiment, the molecule of interest is a TVM of the presentinvention. In another embodiment, the term refers to a T cell withredirected specificity (T-bodies) for a TVM. In another embodiment, anengineered T cell of the present invention expresses a ligand that bindsto or interacts with a TVM. In another embodiment, the engineered T cellexhibits specific activity against a TVC.

In another embodiment, an engineered T cell of the present inventionexpresses a chimeric immunoreceptor (CIR) directed against a TVM. Inanother embodiment, the CIR contains a bi-partite signaling module. Inanother embodiment, the extracellular module of the CIR is a singlechain variable fragment (scFv) antibody that binds or interacts with aTVM. In another embodiment, the intracellular module of the CIR containsa costimulatory domain. In another embodiment, the costimulatory domainis a 4-1BB domain. In another embodiment, the costimulatory domain is aTCR domain. In another embodiment, the CIR contains both a 4-1BB domainand a TCR domain.

In another embodiment, an engineered T cell of the present invention isexpanded in culture. In another embodiment, an engineered T cell of thepresent invention is activated in culture.

Each type of engineered T cell represents a separate embodiment of thepresent invention.

“Cytotoxic virus” refers, in another embodiment, to a virus capable oflysing a cell. In another embodiment, the term refers to a virus capableof lysing a tumor cell. In another embodiment, the virus is arecombinant virus that has been engineered to exhibit a characteristicfavorable for anti-tumor activity. In another embodiment, the virus iswild-type, other than is conjugation to an antibody or ligand of thepresent invention. In another embodiment, the virus is an attenuatedvirus. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the cytotoxic agent or anti-tumor agent isconcentrated in the solid tumor. In another embodiment, the cytotoxicagent or anti-tumor agent is targeted to the solid tumor. In anotherembodiment, concentration of the cytotoxic agent or anti-tumor agentinduces cytotoxicity in a tumor cell of the solid tumor. Eachpossibility represents a separate embodiment of the present invention.

Endothelin antagonists are well known in the art, and are described, forexample, in Dasgupta et al (Dasgupta F, Mukherjee A K, Gangadhar N. CurrMed Chem. 2002 March; 9(5):549-75); Dingemanse et al (Dingemanse J,Clozel M, van Giersbergen P L. J Cardiovasc Pharmacol. 2002 June;39(6):795-802); and Zimmermann et al (Zimmermann M, Seifert V. ClinAuton Res. 2004 June; 14(3):143-5). Each possibility represents aseparate embodiment of the present invention.

The ETRB of methods and compositions of the present invention has, inanother embodiment, the sequence:

MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTPPTKTLWPKGSNASLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEIKETFKYINTVVSCLVFVLGIIGNSTLLRIIYKNKCMRNGPNILIASLALGDLLHIVIDIPINVYKLLAEDWPFGAEMCKLVPFIQKASVGITVLSLCALSIDRYRAVASWSRIKGIGVPKWTAVEIVLIWVVSVVLAVPEAIGFDIITMDYKGSYLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEMLRKKSGMQIALNDHLKQRREVAKTVFCLVLVFALCWLPLHLSRILKLTLYNQNDPNRCELLSFLLVLDYIGINMASLNSCINPIALYLVSKRFKNCFKSCLCCWCQSFEEKQSLEEKQSCLKFKANDHGYDNFR SSNKYSSS(SEQ ID No: 1; GenBank Accession # M74921). In another embodiment, theETRB is a homologue of SEQ ID No: 1. In another embodiment, the ETRB isa variant of SEQ ID No: 1. In another embodiment, the ETRB is an isomerof SEQ ID No: 1. In another embodiment, the ETRB is a proteolyticproduct of SEQ ID No: 1. In another embodiment, the ETRB is a precursorof SEQ ID No: 1. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the ETRB has a sequence set forth in 1 of thefollowing GenBank Accession Numbers: NM_000115, NM_003991, AB209198,E07650, BC014472, 575587, 544866, or 575586. In another embodiment, theETRB is a homologue of 1 of the above GenBank Accession Numbers. Inanother embodiment, the ETRB is a variant of 1 of the above GenBankAccession Numbers. In another embodiment, the ETRB is an isomer of 1 ofthe above GenBank Accession Numbers. In another embodiment, the ETRB isa proteolytic product of 1 of the above GenBank Accession Numbers. Inanother embodiment, the ETRB is a precursor of 1 of the above GenBankAccession Numbers. In another embodiment, the ETRB is encoded by anucleotide sequence set forth in 1 of the above GenBank AccessionNumbers. Each possibility represents a separate embodiment of thepresent invention.

The ET-1 of methods and compositions of the present invention has, inanother embodiment, the sequence:

MDYLLMIFSLLFVACQGAPETAVLGAELSAVGENGGEKPTPSPPWRLRRSKRCSCSSLMDKECVYFCHLDIIWVNTPEHVVPYGLGSPRSKRALENLLPTKATDRENRCQCASQKDKKCWNFCQAGKELRAEDIMEKDWNNHKKGKDCSKLGKKCIYQQLVRGRKIRRSSEEHLRQTRSETMRNSVKSSFHDPKLKGNPSRERYVTHNRAHW (SEQ ID No: 2; GenBank Accession #NM_001955). In another embodiment, the ET-1 is a homologue of SEQ ID No:2. In another embodiment, the ET-1 is a variant of SEQ ID No: 2. Inanother embodiment, the ET-1 is a isomer of SEQ ID No: 2. In anotherembodiment, the ET-1 is a proteolytic product of SEQ ID No: 2. Inanother embodiment, the ET-1 is a precursor of SEQ ID No: 2. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the ET-1 has a sequence set forth in 1 of thefollowing GenBank Accession Numbers: DQ496112, DQ890981, AK226096,BC009720, BC036851. In another embodiment, the ET-1 is a homologue of 1of the above GenBank Accession Numbers. In another embodiment, the ET-1is a variant of 1 of the above GenBank Accession Numbers. In anotherembodiment, the ET-1 is an isomer of 1 of the above GenBank AccessionNumbers. In another embodiment, the ET-1 is a proteolytic product of 1of the above GenBank Accession Numbers. In another embodiment, the ET-1is a precursor of 1 of the above GenBank Accession Numbers. In anotherembodiment, the ET-1 is encoded by a nucleotide sequence set forth in 1of the above GenBank Accession Numbers. Each possibility represents aseparate embodiment of the present invention.

Methods for measuring the expression level of a protein or nucleotide(e.g. mRNA) molecule are well known in the art. In another embodiment,the method comprises a polymerase chain reaction (PCR; see ExperimentalExamples herein). In another embodiment, the method comprises use of anantibody. In another embodiment, the method is Western blotting. Inanother embodiment, the method is an antibody ELISA kit. In anotherembodiment, the method is an RT-PCR kit. In another embodiment, themethod is an RNA isolation kit. In another embodiment, the means is acDNA synthesis kit. In another embodiment, the method is any othermethod of measuring the expression level of a protein or nucleotide thatis known in the art. Each possibility represents a separate embodimentof the present invention.

In another embodiment, a protein or nucleotide molecule of the presentinvention is homologous to a peptide disclosed herein. The terms“homology,” “homologous,” etc, when in reference to any protein orpeptide, refer, in one embodiment, to a percentage of amino acidresidues in the candidate sequence that are identical with the residuesof a corresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Methods and computer programs for the alignment arewell known in the art.

Homology is, in another embodiment, determined by computer algorithm forsequence alignment, by methods well described in the art. For example,computer algorithm analysis of nucleic acid sequence homology caninclude the utilization of any number of software packages available,such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST EnhancedAlignment Utility), GENPEPT and TREMBL packages.

In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 1-90 of greater than 70%. In anotherembodiment, “homology” refers to identity to a sequence selected fromSEQ ID No: 1-90 of greater than 72%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 1-90 of greater than 75%. Inanother embodiment, “homology” refers to identity to a sequence selectedfrom SEQ ID No: 1-90 of greater than 78%. In another embodiment,“homology” refers to identity to one of SEQ ID No: 1-90 of greater than80%. In another embodiment, “homology” refers to identity to one of SEQID No: 1-90 of greater than 82%. In another embodiment, “homology”refers to identity to a sequence selected from SEQ ID No: 1-90 ofgreater than 83%. In another embodiment, “homology” refers to identityto one of SEQ ID No: 1-90 of greater than 85%. In another embodiment,“homology” refers to identity to one of SEQ ID No: 1-90 of greater than87%. In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 1-90 of greater than 88%. In anotherembodiment, “homology” refers to identity to one of SEQ ID No: 1-90 ofgreater than 90%. In another embodiment, “homology” refers to identityto one of SEQ ID No: 1-90 of greater than 92%. In another embodiment,“homology” refers to identity to a sequence selected from SEQ ID No:1-90 of greater than 93%. In another embodiment, “homology” refers toidentity to one of SEQ ID No: 1-90 of greater than 95%. In anotherembodiment, “homology” refers to identity to a sequence selected fromSEQ ID No: 1-90 of greater than 96%. In another embodiment, “homology”refers to identity to one of SEQ ID No: 1-90 of greater than 97%. Inanother embodiment, “homology” refers to identity to one of SEQ ID No:1-90 of greater than 98%. In another embodiment, “homology” refers toidentity to one of SEQ ID No: 1-90 of greater than 99%. In anotherembodiment, “homology” refers to identity to one of SEQ ID No: 1-90 of100%. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, homology is determined via determination ofcandidate sequence hybridization, methods of which are well described inthe art (See, for example, “Nucleic Acid Hybridization” Hames, B. D.,and Higgins S. J., Eds. (1985); Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y). In otherembodiments, methods of hybridization are carried out under moderate tostringent conditions, to the complement of a DNA encoding a nativecaspase peptide. Hybridization conditions being, for example, overnightincubation at 42° C. in a solution comprising: 10-20% formamide, 5×SSC(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA.

Protein and/or peptide homology for any AA sequence listed herein isdetermined, in another embodiment, by methods well described in the art,including immunoblot analysis, or via computer algorithm analysis of AAsequences, utilizing any of a number of software packages available, viaestablished methods. Some of these packages include the FASTA, BLAST,MPsrch or Scanps packages, and, in another embodiment, employ the use ofthe Smith and Waterman algorithms, and/or global/local or BLOCKSalignments for analysis, for example. Each method of determininghomology represents a separate embodiment of the present invention.

In another embodiment of the present invention, “nucleic acids” or“nucleotide” refers to a string of at least two base-sugar-phosphatecombinations. The term includes, in one embodiment, DNA and RNA.“Nucleotides” refers, in one embodiment, to the monomeric units ofnucleic acid polymers. RNA is, in one embodiment, in the form of a tRNA(transfer RNA). In another embodiment, the RNA is snRNA (small nuclearRNA). In another embodiment, the RNA is rRNA (ribosomal RNA). In anotherembodiment, the RNA is mRNA (messenger RNA). In another embodiment, theRNA is antisense RNA. In another embodiment, the RNA is small inhibitoryRNA (siRNA). In another embodiment, the RNA is micro RNA (miRNA). Inanother embodiment, the RNA is a ribozyme. In another embodiment, theRNA is agRNA (antigenic RNA). “agRNA” refers, in another embodiment, toa double stranded RNA capable of interacting with mRNA and silencinggene transcription. The use of siRNA and miRNA has been described (CaudyA A et al, Genes & Devel 16: 2491-96 and references cited therein). DNAcan be, in other embodiments, in form of plasmid DNA, viral DNA, linearDNA, or chromosomal DNA, or derivatives of these groups. In addition,these forms of DNA and RNA can be single, double, triple, or quadruplestranded. The term also includes, in another embodiment, artificialnucleic acids that contain other types of backbones but the same bases.In one embodiment, the artificial nucleic acid is a PNA (peptide nucleicacid). PNA contain peptide backbones and nucleotide bases and are ableto bind, in one embodiment, to both DNA and RNA molecules. In anotherembodiment, the nucleotide is oxetane modified. In another embodiment,the nucleotide is modified by replacement of one or more phosphodiesterbonds with a phosphorothioate bond. In another embodiment, theartificial nucleic acid contains any other variant of the phosphatebackbone of native nucleic acids known in the art. The use ofphosphothiorate nucleic acids and PNA are known to those skilled in theart, and are described in, for example, Neilsen P E, Curr Opin StructBiol 9:353-57; and Raz N K et al Biochem Biophys Res Commun.297:1075-84. The production and use of nucleic acids is known to thoseskilled in art and is described, for example, in Molecular Cloning,(2001, Sambrook and Russell, eds.) and Methods in Enzymology: Methodsfor molecular cloning in eukaryotic cells (2003; Purchio and G. C.Fareed, eds.). Each nucleic acid derivative represents a separateembodiment of the present invention.

In another embodiment, provided herein is a kit comprising a reagentutilized in performing a method of the present invention. In anotherembodiment, provided herein is a kit comprising a composition, tool, orinstrument of the present invention.

“Contacting,” in another embodiment, refers to directly contacting thetarget cell with a composition of the present invention. In anotherembodiment, “contacting” refers to indirectly contacting the target cellwith a composition of the present invention. Each possibility representsa separate embodiment of the present invention. In another embodiment,the composition of the present invention is carried in the subjects'bloodstream to the target cell. In another embodiment, the compositionis carried by diffusion to the target cell. In another embodiment, thecomposition is carried by active transport to the target cell. Inanother embodiment, the composition is administered to the subject insuch a way that it directly contacts the target cell. Each possibilityrepresents a separate embodiment of the present invention.

Pharmaceutical Compositions and Methods of Administration

Pharmaceutical compositions containing compositions of the presentinvention can be, in another embodiment, administered to a subject byany method known to a person skilled in the art, such as parenterally,paracancerally, transmuco sally, transdermally, intramuscularly,intravenously, intra-dermally, subcutaneously, intra-peritonealy,intra-ventricularly, intra-cranially, intra-vaginally orintra-tumorally.

In another embodiment of methods and compositions of the presentinvention, the pharmaceutical compositions are administered orally, andare thus formulated in a form suitable for oral administration, i.e. asa solid or a liquid preparation. Suitable solid oral formulationsinclude tablets, capsules, pills, granules, pellets and the like.Suitable liquid oral formulations include solutions, suspensions,dispersions, emulsions, oils and the like. In another embodiment of thepresent invention, the active ingredient is formulated in a capsule. Inaccordance with this embodiment, the compositions of the presentinvention comprise, in addition to the active compound and the inertcarrier or diluent, a hard gelating capsule.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intra-muscular injection of a liquidpreparation. Suitable liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In anotherembodiment, the pharmaceutical compositions are administeredintravenously and are thus formulated in a form suitable for intravenousadministration. In another embodiment, the pharmaceutical compositionsare administered intra-arterially and are thus formulated in a formsuitable for intra-arterial administration. In another embodiment, thepharmaceutical compositions are administered intra-muscularly and arethus formulated in a form suitable for intra-muscular administration.

In another embodiment, the pharmaceutical compositions are administeredtopically to body surfaces and are thus formulated in a form suitablefor topical administration. Suitable topical formulations include gels,ointments, creams, lotions, drops and the like. In another embodiment,for topical administration, the compositions are prepared and applied assolutions, suspensions, or emulsions in a physiologically acceptablediluent with or without a pharmaceutical carrier.

In another embodiment, the active compound is delivered in a vesicle,e.g. a liposome.

In other embodiments, carriers or diluents used in methods of thepresent invention include, but are not limited to, a gum, a starch (e.g.corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol,sucrose, dextrose), a cellulosic material (e.g. microcrystallinecellulose), an acrylate (e.g. polymethylacrylate), calcium carbonate,magnesium oxide, talc, or mixtures thereof.

In other embodiments, pharmaceutically acceptable carriers for liquidformulations are aqueous or non-aqueous solutions, suspensions,emulsions or oils. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,emulsions or suspensions, including saline and buffered media. Examplesof oils are those of animal, vegetable, or synthetic origin, forexample, peanut oil, soybean oil, olive oil, sunflower oil, fish-liveroil, another marine oil, or a lipid from milk or eggs.

In another embodiment, parenteral vehicles (for subcutaneous,intravenous, intraarterial, or intramuscular injection) include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's and fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers such as those based onRinger's dextrose, and the like. Examples are sterile liquids such aswater and oils, with or without the addition of a surfactant and otherpharmaceutically acceptable adjuvants. In general, water, saline,aqueous dextrose and related sugar solutions, and glycols such aspropylene glycols or polyethylene glycol are preferred liquid carriers,particularly for injectable solutions. Examples of oils are those ofanimal, vegetable, or synthetic origin, for example, peanut oil, soybeanoil, olive oil, sunflower oil, fish-liver oil, another marine oil, or alipid from milk or eggs.

In other embodiments, the compositions further comprises binders (e.g.acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g. cornstarch, potato starch, alginic acid,silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodiumstarch glycolate), buffers (e.g., Tris-HCI, acetate, phosphate) ofvarious pH and ionic strength, additives such as albumin or gelatin toprevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g.sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g.,glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosityincreasing agents (e.g. carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g. aspartame, citric acid),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g. stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g. colloidal silicon dioxide),plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers(e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymercoatings (e.g., poloxamers or poloxamines), coating and film formingagents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/oradjuvants. Each of the above excipients represents a separate embodimentof the present invention.

The compositions also include, in another embodiment, incorporation ofthe active material into or onto particulate preparations of polymericcompounds such as polylactic acid, polyglycolic acid, hydrogels, etc, oronto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts.) Such compositionsinfluence, in another embodiment, the physical state, solubility,stability, rate of in vivo release, and rate of in vivo clearance.

The preparation of pharmaceutical compositions that contain an activecomponent, for example by mixing, granulating, or tablet-formingprocesses, is well understood in the art. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active agents are mixed with additives customary forthis purpose, such as vehicles, stabilizers, or inert diluents, andconverted by customary methods into suitable forms for administration,such as tablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic or oily solutions. For parenteral administration, the activeagents are converted into a solution, suspension, or emulsion, ifdesired with the substances customary and suitable for this purpose, forexample, solubilizers or other substances.

Each of the above additives, excipients, formulations and methods ofadministration represents a separate embodiment of the presentinvention.

In one embodiment, the term “administering” refers to bringing a subjectin contact with an active compound of the present invention. In anotherembodiment, administration is accomplished in vitro, i.e. in a testtube. In another embodiment, administration is accomplished in vivo,i.e. in cells or tissues of a living organism. Each possibilityrepresents a separate embodiment of the present invention.

In one embodiment, the methods of the present invention compriseadministering an active composition or compound of the present inventionas the sole active ingredient. However, also encompassed within thescope of the present invention are methods for chemotherapy thatcomprise administering the active composition or compound in combinationwith one or more therapeutic agents (e.g. anti-tumor agents or cancerchemotherapy agents).

Experimental Details Section Materials and Experimental Methods Tissues

Stage-III epithelial ovarian cancer and ductal breast cancer specimenswere collected at the University of Turin, Italy, following informedconsent, from previously untreated patients. Additional ovarian cancerspecimens, and normal ovaries were collected at the University ofPennsylvania Medical Center after obtaining written informed consentunder Institutional Review Board (IRB)-approved protocols. Malignantmesothelioma (n=3), non-small cell lung carcinoma (n=3) (provided by Dr.Steven M. Albelda) and malignant melanoma (n=3) (provided by Dr. DavidElder) were collected after obtaining written informed consent underIRB-approved protocols. A panel of normal human tissues (FIG. 3) wasprovided by the Cooperative Human Tissue Network. All specimens wereprocessed in compliance with HIPAA requirements.

Reagents

Antibody against human CD31 (BD Pharmingen) followed by secondaryantibodies (Vector, Burlingame, Calif.) were diluted (1:10) in PBScontaining RNA Protector (1:10, Roche, Basel, Switzerland). Streptavidinconjugate and AEC chromagen (Dako, Carpenteria, Calif.) were diluted inPBS containing RNA Protector. Laser Capture Microdissection (LCM) wasperformed using Microcut (MMI, Glattbrugg, Switzerland), employing lessthan three hours per slide.

RNA Isolation

In order to increase RNA yield, dissected samples were treated withpre-digested proteinase-K. RNA was isolated using TRIzol reagentmicroprotocol (Gibco, Carlsbad, Calif.). Glycogen carrier (20 μg) wasutilized to increase RNA yield in all protocols. RNA integrity andquantity were assayed using the Bioanalyzer (Agilent, Foster City,Calif.).

RNA Amplification

RNA was amplified using Messageamp® (Ambion, Austin, Tex.), with thefollowing modifications: First-strand synthesis was performed at 42° C.(2 hours), then 48° C. (10 min). After second-strand synthesis, RNA wastranscribed at 37° C. (12 hours); T7-polymerase and RNAse inhibitor wereadded and transcription was continued for 12 more hours. After 2 roundsof amplification, cRNA was biotin-labeled (12-24 hours, ENZO RNAlabeling kit, Farmingdale, N.Y.) and purified using RNA cleanup (Qiagen,Valencia, Calif.).

Arrays

Immunohistochemistry-guided laser capture microdissection was performedfrom 24 epithelial ovarian cancers (EOC) with or without (12 each)intratumoral T cells (ITC). CD31 positive cells with a vascularmorphology were isolated and RNA extracted using TRIzol. RNA wasamplified using the Ambion MessageAmp kit, and hybridized to the U133aand U133B human genome arrays from Affymetrix.

Array Analysis

Genes were identified that were present in at least 1 of the 29 samplesanalyzed; and only those genes that demonstrated at least a 1.5-foldincrease or decrease in relative expression between ITC(+) and ITC(−)tumor vascular cells were further analyzed. Using hierarchicalclustering, a gene tree was generated using the resulting list ofdifferentially genes. Molecules were identified that were present invascular cells from at least 9 of 14 ITC(+) tumors and upregulated by atleast 2-fold compared to ITC(−) vascular cells. Similarly, moleculeswere identified that were present in vascular cells from at least 6 of11 ITC (−) tumors and 2-fold upregulated compared to ITC(+) tumorvascular cells using Genespring software (Agilent Technologies, SantaClara, Calif.). Quantitative PCR (qPCR) and Western blot of 60 EOCtumors was used to confirm over-expression of Endothelin B receptor(ETRB) in ITC(−) tumors.

qRT-PCR

qRT-PCR was performed using primers to the 3′ end of transcriptsspanning intron-exon boundaries whenever possible for 35 cycles usingSybergreen® (ABI, Foster City, Calif.), with primers at 150 nMconcentrations. Primers were 18-24 nucleotides and were designed to havea TM of 59-61° C. All transcripts were confirmed using 3% agarose gelelectrophoresis. Gene expression was normalized against β-actin in allstudies unless stated otherwise.

Immunostaining

For validation studies, immunohistochemistry (IHC) was performed usingthe VECTASTAIN ABC® kit (Vector, Burlingame, Calif.). All primaryantibodies were incubated for one hour. Immuno-reaction was visualizedwith 3,3′-diaminobenzidine (Vector). All staining steps were performedat room temperature.

Bioinformatic and Statistical Analyses

Statistical significance for mRNA expression differences between tissuetypes was determined using a two-tailed Student's t-Test. Pearson'scorrelation was used to determine linearity of arrays performed with oneversus two rounds of amplification or before and after immuno-LCM.Analyses of expression profiles were performed using Genespring software(Agilent); all samples were normalized with the median defined as 1.0. Aheat map condition tree was developed using a hierarchical clusteringalgorithm (Genespring®) excluding all genes where the difference betweenthe means of the tumor and normal vascular samples was less than itsstandard error. Descriptive statistics were performed with the SPSS®statistics software package (SPSS, Ill., USA). The algorithm for thenonparametric method based on the ranks of the expression level fortumor and normal samples was developed in SAS 9.1.

Optimization of Immunostaining

To procure highly purified tumor vasculature, a rapid and reliableimmuno-LCM protocol was established for microarray applications.Different fixation conditions were tested, including −20° C. acetone;70% ethanol: 10% acetic acid (1:1 vol:vol); methanol; or 4%paraformaldehyde. Fixation with acetone or ethanol-based fixativesresulted in the greatest RNA yield (FIG. 2A). Acetic acid:ethanolfixation did not enable optimal IHC visualization of select targetproteins. Acetone fixation was chosen for all further experiments.

Next, immunostaining was optimized. RNA isolated from tissue sectionsafter standard IHC using LSAB (Dako) or Vectastain (Vector) showednear-complete degradation (FIG. 2B-6). We thus developed an ultra-fastIHC protocol with increased concentrations of reagents. Highconcentrations of RNAse inhibitor were added to all aqueous solutions.The choice of RNAse inhibitor was critical for RNA integrity. RNAProtector® (Roche) was found to be superior to placental RNAse inhibitor(Stratagene) or SuperRNAsin® (Ambion), leading to two-fold increase inRNA yield and integrity (FIG. 2B). Combining RNAse inhibitors reducedthe efficacy of RNA Protector. Addition of RNA Protector to IHC allowedfor 90% preservation of RNA integrity, based upon comparison ofribosomal RNA ratios determined using the Agilent Bioanalyzer.

Next detection systems were compared. AEC chromagen resulted in 40%greater RNA yield than DAB. Immunofluorescence resulted in 100%increased RNA yield compared to AEC (FIG. 1C), but contaminating cellswere poorly identifiable without counterstaining, as assessed by qRT-PCRdetection of the T-cell marker CD3-ε. Furthermore, fluorescencequenching limited LCM time. Thus, AEC IHC was used for subsequentexperiments.

In addition, the effect of LCM time on RNA yield and integrity wasexamined. Leaving immunostained tissue sections at room temperature forup to three hours before RNA was isolated had no significant impact onthe quality or quantity of RNA isolated (FIG. 1D).

Optimization of RNA Purification.

RNA amplification methods (Arcturus Picopure kit, Stratagene microRNAisolation kit, Zymo mini RNA isolation kit and the modified TRIzolmethod for less than 10⁵ cells) were compared for RNA yield and qualityafter immuno-LCM. Arcturus Picopure gave the highest RNA yield fortissues stained with hematoxylin alone, but not following IHC (FIG. 2E).The protocol from ZYMO also resulted in low RNA yield. Conversely, theStratagene micro RNA isolation kit and the modified TRIzol method gavesignificantly better and similar yields by quantification with theAgilent Bioanalyzer.

RNA quality was tested by qRT-PCR of GADPH and β-actin transcripts intotal RNA procured from 1×10⁶ cells microdissected and processed as inTable 2. GADPH or β-actin transcripts were detected at similar levels inRNA isolated with the modified TRIzol method using phase-lock tubes(Eppendorf, Hamburg, Germany) or with the Stratagene micro RNA isolationkit. Arcturus picopure and ZYMO RNA isolation kits were 10-fold and256-fold less sensitive, respectively (FIG. 2F).

The resulting protocol, requiring approximately 25 minutes for IHC,proved successful for numerous antibodies (Table 1). While someantibodies required longer incubation times (up to 15 minutes), therewas no loss of RNA yield or integrity. Staining was quite specific, evenwith high concentrations of antibody. The protocol was reproducibly ableto capture 500,000 μm² of tumor vascular cells in 3 hours of microdissection and recover ˜20 ng total RNA. RNA was reproducibly amplified to15 μg of biotin-labeled cRNA.

Optimization of RNA Amplification

The linearity of amplifications using Ambion MessageAmp® was tested bycomparing the gene expression profile of 10 μg unamplified whole tumorRNA to amplified 6, 24 or 60 ng of the same RNA. Transcriptionalprofiling was performed using Affymetrix U133 chips. Correlation betweenunamplified RNA and 24 or 60 ng input RNA was high (r²=0.93 and 0.94,respectively) (FIG. 2G). Correlation was lower with 6 ng input RNA(r²=0.87). High correlation was found between gene expression profilesfrom amplifications of input. RNA procured from the same tumor performedwithin the same experiment (intra-assay, r²=0.99) or in differentexperiments (inter-assay, r²=0.97). Immuno-LCM had no impact onexpression profile (FIG. 1C).

TABLE 1 List of antibodies tested, company and clone used in the studyas indicated. Success with staining using the rapid IHC protocol for LCMfollowing fixation in acetone or acetic acid/ethanol (AA/EtOH) isreported on the side for tested antibodies. AA/ Antibody Company CloneAcetone EtOH Biot hCD45 BD Pharm H130 *** — Biot hCD31 Ancell 158-2B3*** ** Biot hCD31 Caltag MBC 7B.2 ** ** hCD31 Dako JC70A ** ** BiotCD146 Chemicon MAB16985B *** *** CytoKeratin Dako AO575 * — Biot hCD3 BDPharm UCHT1 *** — Fitc hCD31 BD Pharm WM59 *** *** SMA-α-Cy3 Sigma 1A4ND ND FOLH1 Zymed ZMD.80 ND ND STC2 Genway A22017 ND ND Biot CD74 BDPharm Mb741 ND ND AML-1 Active Motif Polyclonal ND ND hCD34-PE BD Pharm581 ND ND F-Spon Abcam Ab14271-50 ND ND Lrp4 Abcam Ab13388-25 ND NDEndothelial Cayman Chemical Polyclonal ND ND Lipase (—), poor stain, (*)fair stain, (**) good stain, (***) excellent stain. ND, not determined.

The optimized Immuno-LCM protocol is summarized in Table 2.

TABLE 2 Summary of Immuno-LCM Protocol Tumor Freshly cut 8 μm sectionsof snap frozen tumor IHC** Fix in −20° C. Acetone - 4 min Incubate withprimary Ab 1:10 - 5 min Incubate with 3x biotinylated anti-mouse Ab(Vector) - 5 min. Brief wash in PBS 2.5X Streptavidin-biotinamplification (DAKO) - 5 min Brief wash in PBS AEC (DAKO) stain - 3 to 5min Brief wash in PBS Stain with dilute hematoxylin Rinse (** All stepswith 1:10 RNAse Protector) LCM Dry tissue sections with hair dryer - 1min Microdissect cells - up to 3 hours RNA isolation Treat withProteinase K (10 μg/ml) - 8 min Extract RNA with TRIzol in phase lockgel - 1 hour RNA Amplification Use Ambion MessageAmp ®

During the optimization of RNA isolation and amplification methodology,it was found that the immuno-LCM procedure had minimal impact on RNAintegrity (FIG. 2A-F) or gene expression profiling (FIGS. 1C and 2G).

The absence of tumor cell and lymphocyte lineage-specific markers wasconfirmed in immuno-LCM purified TECs by RT-PCR and quantitativereal-time polymerase chain reaction (qRT-PCR).

ETRB as an Ovarian Carcinoma Biomarker

RNA was isolated from 61 snap-frozen advanced stage (III and IV) EOCspecimens collected from previously untreated patients undergoingdebulking surgery. Quantitative PCR was used to assay ETRB expression.The Wilcoxon rank-sum test was used to compare ENDR expression amonggroups defined by ITC and debulking. The survival curves were estimatedusing the Kaplan-Meier procedure. Hazard ratios for ENDR expression wereobtained from Cox proportional hazard models and presented with their95% confidence intervals.

BQ788 as a Tumor Vaccine Adjuvant

Two injections of 5×10 6^(th) UV irradiated ID8 ovarian cancer cellswere injected subcutaneously in C57B16 mice one week apart. Vaccinatedmice and non-vaccinated controls were injected with 5×10⁶ ID8 cells inthe flank with 300 ml matrigel or intraperitoneally. Tumors were allowedto grow for 2 or 5 weeks as indicated, and then treated withintraperitoneal injections of BQ-788 (300 mcg) or control peptide for 2weeks.

FACS analysis was performed using APC-CD45 (BD Pharmingen), PE-anti CD3,FITC anti CD4, and Biotin anti-CD8 coupled with streptavid PE-Cy7.

IHC was performed using the Vectastain kit (Vector) mouse anti-ETRB(Abcam 1922-225), anti ADRBK2 (AbCAM, rabbit polyclonal), anti-ESRalpha(Genetex 1D5). Western blots were performed using the anti-ETRB 1:200and HRP anti-rabbit secondary (Santa Cruz Biotechnology).

Cell Culture

Human vascular endothelial cells (HUVEC) were grown to 70% confluence inEBM media then treated with 50 nM Endothelin receptor B inhibitor BQ-788(American peptide), or 2.5 nm Endothelin alone or in the presence ofeither 50 nm Endothelin receptor A inhibitor BQ123 (American peptide),or 50 nM Endothelin receptor B inhibitor BQ-788. Alternatively cellswere treated with BQ-788 alone. Media was changed every 48 hrs for atotal of 6 days. After 6 days cells were harvested for RNA, flowcytometry or incubated activated T cells. T cells were activated for 48hours with either 5 ng/ml PMA (ref) or CD3 and CD28 beads. Afteractivation T cells were labeled with CFSE and then incubated withpretreated endothelial cells for 2 hours with shaking. Cells were thenwashed 3× with PBS and adhesions was determined using a fluorescentplate reader.

Example 1: Identification of Distinct Endothelial Profiles in TumorsContaining or Lacking Intraepithelial T Cells

Immunohistochemistry-guided laser capture microdissection (immuno-LCM)coupled with RNA amplification and genome-wide transcriptional profilingwas utilized to analyze high-quality RNA from highly purified tumorendothelial cells. In preparatory experiments, 21 tumor endothelialcells (TEC) and 4 normal ovarian endothelial cell (EC) specimens wereanalyzed and to identify genes that are specifically expressed in tumorendothelium. In the present experiment, TEC samples were divided intoovarian tumors with brisk intraepithelial (IE) T cell (n=14) and tumorslacking altogether IE T cells (n=11), and unsupervised hierarchicalclustering was performed using 17,920 genes (after elimination of allgenes wherein the difference between TEC and normal endothelial cellmeans was less than the standard error of the difference in the means).TECs of tumors with IE T cells were accurately classified from TECs oftumors lacking IE T cells, demonstrating a clear difference in molecularprofiles (FIG. 3). When unsupervised hierarchical clustering includedalso profiles of normal EC, TEC from tumors lacking IE T cells clusteredclosely with normal EC.

Among genes differentially expressed (>2.5-fold) between the 2 types ofTEC (FIG. 3), genes that were upregulated in TEC from tumors lacking IET cells included the endothelin receptor B (ETRB); the RNA bindingprotein homolog Musashi 2 (MSI2); and 2 members of the Notch signalingpathway, delta-like 1 and Hairy/Enhancer of Split 1, while genes thatwere upregulated in TECs of tumors harboring IE T cells included thecomplement component 3 (C3); the apoptosis regulator CFLAR; the estrogenreceptor alpha (ESR1); and the adrenergic receptor B2 (ADRBK2). Thus,expression profiling distinguished TECs from tumors with or without IE Tcells and identified TEC molecules specifically associated with theabsence of IE T cells.

The genes identified are set forth in Table 3:

Fold change Common name and/or Gene Symbol GenBank Accession Number/SEQID Number Genes upregulated in ITC⁻ TVC 3.627 MEGS (Maternally expressed3) AI291123; AB032607; 3-4 BC036882; BC036882; BC062783; AJ413186;AK055725; AK057522; AK092504; AK092707; AK124580; AK127864 2.886 SEC61G(Sec61 gamma subunit) NM_014302; BC009480;  5 BC051840; NM_014302;AF086539; 2.873 KIAA1609 AA195124; BC023251 6-7 2.82 ACTR6 (ARP6actin-related NM_022496; BC015107;  8 protein 6 homolog) AB038229;AF212251; AK023495; AK023684; AK124075 2.784 FLJ23006 fis, cloneLNG00414 AK026659  9 2.746 ATP9A (ATPase, Class II, type AB014511;AF086357; 10-11 9A) AK025559; BC016044; BC036759; AB014511; NM_0060452.665 IMAGE: 23539 R38110 12 2.642 NCOA1 (Nuclear receptor BF576458;AJ000881; AJ000882; 13-14 coactivator 1) U59302 2.584 Wilms tumorupstream neighbor 1 NM_015855; BC002734 15 (WIT1) 2.539 IMAGE: 1909757AI343000 16 2.513 MSI2 (Musashi homolog 2) BE220026; BC017560; 17-18AK093888 2.502 ETRB NM_000115; AB209198; 19 D90402; S57283 2.473 PAPSS2(3′-phosphoadenosine 5′- AW299958; AF091242 20-21 phosphosulfatesynthase 2) 2.372 aldolase A, fructose-bisphosphate NM_000034 22 (ALDOA)2.372 ZNF423 (Zinc finger protein 423) AW149417; NM_015069 23-24 2.358ENPP2 (Ectonucleotide L35594; BC034961; AK124910; 25pyrophosphatase/phosphodiesterase AK130313; D45421; 2 (autotaxin))NM_001040092; NM_006209 2.344 HSU79266 (a.k.a. SAC3D1; SAC3 NM_013299;BC007448; U79266 26 domain containing 1) 2.34 KIAA0146 D63480 27 2.316IMAGE: 1902075 AI300126 28 2.279 EMX2 (Empty spiracles homolog AI478455;NM_004098; 29-30 2) AF301598; BC010043; AK055041 2.273 MYBL1; (V-mybmyeloblastosis AW592266; X66087 31-32 viral oncogene homolog (avian)-like 1) 2.27 MPHOSPH9 X98258 33 2.267 IMAGE: 1660792 AI083578 34 2.233ETRB M74921 35 2.214 IMAGE: 191524 H37807 36 2.212 IMAGE: 2365035AI800895 37 2.17 TAF3 (TAF3 RNA polymerase II, AI123516; AL117661;BC028077 38-39 TATA box binding protein (TBP)- BC062352 associatedfactor, 140 kDa) 2.148 SLC1A4 (Solute carrier family 1 BF340083;BC026216; 40-41 (glutamate/neutral amino acid NM_003038 transporter),member 4) 2.141 HES1 (Hairy and enhancer of split BE973687; BC039152;42-43 1) NM_005524; AF264785; AK000415 2.135 DLK1 (Delta-like 1 homolog)U15979; BC007741; BC013197; 44 BC014015; NM_001032997; NM_003836 2.122SGCB (Sarcoglycan, beta (43 kDa U29586; BC020709 45dystrophin-associated glycoprotein) Genes upregulated in ITC⁺ TVC 5.412complement component 3 (C3) NM_000064 46 3.746 IMAGE: 2755380 AW26231147 3.455 ZNFN1A5 (a.k.a. IKZF5 (IKAROS BF056303; AK023288; 48-49 familyzinc finger 5 (Pegasus)) AK055507 3.141 LOC283663 AI926479; AL713736;50-51 AK090515; AK097083; AK123700 3.096 IGLJ3 (Human rearranged X57812;BC012159; BC015833; 52 immunoglobulin lambda light BC018749; BC020233;chain mRNA) BC020236; BC022098; BC022823 2.872 ZNF521 (Zinc fingerprotein 521), AK021452; AL117615; 53 BC032869 2.831 clone COL05405AK000119 54 2.682 CALD1 (Caldesmon 1) BF063186; BC040354; 55-56NM_004342; NM_033138-140; AB062484; AJ223812; BC015839; BX538339;BX648808 2.678 cytochrome P450, family 1, NM_000104; NM_000104; 57subfamily B, polypeptide 1 U03688 (CYP1B1) 2.65 EIF5B (Eukaryotictranslation BG261322; BC032639; 58-59 initiation factor 5B) AJ006412;AL133563; AB018284; AJ006776; AK091864; NM_015904 2.618 IMAGE: 1518332AA903710 60 2.587 HSPC056 (a.k.a. ARMC8; BF942281; BC032661; 61-62Armadillo repeat containing 8), BC041699 2.576 FLJ32949 (a.k.a. DPY19L2(Dpy- AI039361; AL833344; 63-64 19-like 2 (C. elegans)) NM_173812;AY358792 2.48 CFLAR (CASP8 and FADD-like AI634046; Y14040; AF00577565-66 apoptosis regulator) 2.467 IMAGE: 244300 N54783 67 2.457FLJ10330/PRPF38B (PRP38 pre- N32872; BC007757; BC009453; 68-69 mRNAprocessing factor 38 (yeast) BC040127; BC107801 domain containing B)2.455 C18orf14 NM_024781; BC007757; 70 BC009453; BC040127; BC107801 2.45IMAGE: 2115041 AI417595 71 2.448 GBP1/GBP3 (Guanylate binding AW014593;AB208912; 72-73 protein 3) M55542; NM_002053 2.438 IMAGE: 731714AA417078 74 2.427 SFRS1 (Splicing factor, AA046439; BC010264; 75-76arginine/serine-rich 1 (splicing NM_006924; AB062124; factor 2,alternate splicing factor)), AB209558 2.426 NICAL; MICAL1 (MicrotubuleNM_022765; BC009972; 77 associated monoxygenase, calponin BC042144;BC052983; and LIM domain containing 1) AB048948; AK025392; BC036514;AK021999; AK024500; AK160384 2.419 NOL7 NM_016167; BC062683; 78BC023517; AF172066 2.41 MYCBP2 (MYC binding protein 2) AA488899;AF075587; 79-80 BX647202; AB020723; AK092651; NM_015057 2.382 estrogenreceptor 1 (ESR1) NM_000125 81 2.382 IMAGE: 2275600 AI683805 82 2.356ADRBK2 (Adrenergic, beta, AI651212; BC029563; 83-84 receptor kinase 2)BC063545; AK055687; AK123767 2.348 EST366269 MAGE resequences AW95419985 2.346 SCAP2/SKAP2 (Src kinase NM_003930; BC036044 86 associatedphosphoprotein 2) 2.328 Homo sapiens serine/threonine NM_006281;BC010640; 87 kinase 3 (STE20 homolog, yeast) AK131363; U26424 (STK3)2.324 AKAP10 (A kinase (PRKA) AU147278; BC017055; 88-89 anchor protein10)

Example 2: Validation of Endothelial Genes Associated with IE T-Cells

All of the above genes were detected in whole tumor RNA from a differentset of tumors (n=28) (FIG. 4A) as well as in CD146⁺ VE-cadherin⁺ CD45⁻TEC freshly immuno-purified by FACS from advanced ovarian cancers (n=7).Overexpression of ETRB, KIAA1609, and NCOA in tumors lacking IE T cells(n=12) was confirmed by qRT-PCR (4.3-fold and 2.2-fold respectively,p<0.05). Furthermore, ETRB, KIAA1609, and NCOA were significantlyoverexpressed by qRT-PCR in TEC from tumors lacking IE T cells (all,p<0.0x; n=3) (FIG. 4). Furthermore, overexpression of C3, caldesmin-1,HSPC056, ADRBK2, and ESR1 in tumors exhibiting IE T cells was confirmedby qRT-PCR (all p<0.05; n=16) (FIG. 4A). C3, caldesmin-1, HSPC056,ADRBK2, and ESR1 were significantly overexpressed by qRT-PCR also in TECfrom tumors harboring IE T cells (t-test; n=4) (FIG. 4B). Thus,association of specific endothelial genes with the presence or absenceof IE T cells was confirmed by qRT-PCR.

Example 3: Overexpression of ETRB and its Ligand, ET-1, Associate withLack of IE T-Cells

ETRB was consistently associated with absence of IE T cells in humanovarian cancer; thus, expression of this protein in ovarian cancer andits function in T cell homing were examined further. Consistent with theresults above, ETRB protein was detected by IHC in ovarian tumorvasculature and stromal cells, but not in tumor cells. IHC revealedhigher expression of endothelial ETRB in tumors lacking IE T cellsrelative to tumors harboring T cells. The endothelial location of ESR1and ADRBK2 was validated by IHC with available antibodies.

ETRB protein was further quantified by Western blotting in ovariancancer samples (n=40); it was detected at lower levels in the 20 tumorsharboring IE T cells, but was robustly expressed in 16 of 20 tumorslacking IE T cells (FIG. 5). Among tumors with IE T cells, thoseexpressing ETRB were associated with lower density of IE T cellscompared to tumors lacking IE T cells as assessed by CD3 IHC as well asCD3-epsilon mRNA levels. Thus, increased expression of ETRB by tumorendothelium is associated with absence or paucity of IE T cells.

Expression of the ligand of ETRB, endothelin-1 (ET-1), was examined inovarian cancer. ET-1 expression was restricted to tumor islets. To testwhether ET-1 is expressed by tumor cells, ET-1 mRNA levels in highlypurified tumor cells procured by immuno-LCM were quantified. Strongexpression of ET-1 in vivo was documented in tumor cells isolated from10 ovarian cancers. Further, ET-1 expression was significantly higher intumors lacking IE T cells relative to tumors harboring IE T cells.Collectively, these data show that over-expression of ETRB by tumorendothelium and its ligand ET-1 by tumor cells is associated withabrogation of T cell infiltration in tumor islets. Further, thesefindings show that a molecular crosstalk occurs between tumor cells andtumor endothelium that predicts lack of T cell homing to tumors and showan important role of the ET-1/ETRB axis in controlling T celltrafficking in tumors.

Example 4: ETRB Overexpression Predicts Poor Outcome in Ovarian Cancer

IE T cell infiltration is a strong predictor of clinical outcome inovarian cancer. To determine whether ETRB overexpression is predictiveof poor outcome in ovarian cancer, ETRB expression was quantified byqRT-PCR in 62 EOC specimens (38 with and 23 lacking IE T-cells) andpatients were stratified into groups. There were significant differencesin the distributions of both overall survival and disease-free survival,according to high and low expression of ETRB (p<0.001); the five-yearoverall survival rate was 41% for patients whose tumors exhibited higherETRB expression versus 100% for those whose tumors exhibited the lowestexpression ETRB EOC patients (FIG. 6). In univariate analysis, thehazard ratio for lowest ETRB-expressing group was 0.05 for overallsurvival (95% CI 0-0.42, p<=0.005) and 0.15 for disease-free survivalcompared to the highest group (95% CI 0.04-0.56, p<=0.005). Highexpression of ETRB strongly correlated with absence of IE T-cells.

Example 5: Endothelial ETRB Regulates T Cell Trafficking Materials andExperimental Methods

The murine epithelial ovarian cancer cell line ID8, syngeneic to C57BL/6mice was cultured in DMEM supplemented with 4% FBS, 13 ITS mediasupplement (bovine insulin (5 mg/L), human transferrin (5 mg/L), andsodium selenite (5 mg/L); Sigma), and antibiotics.

Flank and Orthotopic, Intraperitoneal ID8 Models

Female C57BL/6 mice (8 weeks of age) were injected 3 times i.p. with1×10⁶ UV-treated, apoptotic ID8 ovarian cancer cells (resuspended in 500microliter (mcL) DMEM without supplements) in the flank. For theorthotopic, intraperitoneal model, mice were injected i.p. with 5×10⁶ID8 cells.

Results

To confirm that ETRB plays a role in inhibiting T cell homing to humanovarian cancers, the ID8 syngeneic mouse model of ovarian cancer wasutilized. This model responds modestly to potent dendritic cell (DC)vaccination. Strong expression of ETRB was detected in tumor endotheliumin ID8 flank tumors by IHC (FIG. 7). Mice were vaccinated with asuboptimal preventive vaccine, containing UV-treated ID8 cells, whichresults in induction of systemic tumor-reactive interferon-gammasecreting T cells without significant delay in tumor growth Followingvaccination, mice were inoculated with flank tumors, which were allowedto engraft for 2 or 5 weeks, and then mice were treated i.p. with theETRB antagonist peptide, BQ-788, SKRGRRPGAKALSRVREDIVE (SEQ ID No: 90),every 2^(nd) day for 2 weeks or with control peptide that was ascrambled version of the above peptide. Additional controls includednon-vaccinated animals treated with BQ-788 or control peptide. Toconfirm that the above vaccination scheme results in significantincrease in the frequency of systemic tumor-reactive T cells, CD3⁺/CD8⁺splenocytes from vaccinated animals treated with BQ-788 or controlpeptide, and from non-vaccinated mice, were labeled with CFSE andincubated with DC pulsed with UV-radiated ID8 for 6 days to assessproliferation. T cells from non-vaccinated animals showed minimalproliferation, while T cells from vaccinated animals exhibited robustproliferation, confirming the presence of anti-tumor T cells in theseanimals (FIG. 8C). Proliferation of lymphocytes from vaccinated micetreated with BQ-788 or control peptide was similar. Similarly, in CTLassays, CD3⁺/CD8⁺ splenocytes from vaccinated animals treated withBQ-788 or control peptide exhibited robust ID8 cell killing, whileCD3⁺/CD8⁺ splenocytes from non-immunized mice exhibited no killing (FIG.8D).

Treatment of vaccinated mice with BQ-788, starting at 2 or at 5 weeks,led to significant reduction in tumor growth (FIG. 6 B, FIG. 7). Tumorgrowth delay was not observed in non-vaccinated mice treated with BQ-788or in vaccinated mice treated with control peptide. Tumors fromvaccinated mice treated with BQ-788 exhibited areas with very stronginfiltration by CD8⁺ T cells. In contrast, non-vaccinated animalstreated with BQ-788 as well as vaccinated animals treated with controlpeptide exhibited scarce intratumoral CD8⁺ T cells (FIG. 8). Flowcytometry from mechanically dissected tumors confirmed the resultsobserved with IHC: In non-vaccinated animals treated with BQ-788 as wellas in vaccinated animals treated with control peptide, CD3⁺ cellsrepresented on average 4% of the cells (range 0.5 to 12%), whilevaccinated animals treated with BQ-788, CD3⁺ cells represented 15% ofthe cells (range 8 to 30%), containing both CD4⁺ and CD8⁺ cells (FIG.8).

The impact of BQ-788 on survival in vaccinated animals was also testedin the orthotopic, intraperitoneal ID8 model of ovarian cancer.Following vaccination, mice were injected i.p. with ID8 cells. Two weekslater, animals received either BQ-788 or control peptide every 2^(nd)day for 2 weeks. Vaccinated animals treated with BQ-788 developedascites later than vaccinated animals treated with control peptide andexhibited a significant prolongation of survival (FIG. 8F). Thus,systemic administration of an ETRB antagonist markedly enhances theability of effector cells, previously induced through vaccination, tohome to tumors and exert rejection.

Example 6: ETRB Blockade Upregulates Endothelial ICAM-1

Next, the effect of BQ-788 on human and murine endothelial cells or Tcells was tested in the presence or absence of ET-1 ligand. In addition,the effect of endothelin receptor A antagonist BQ123 was tested.Treatment of HUVEC with BQ-788 in the presence of Endothelin led to adistinct morphological change in the HUVEC cells (FIG. 9). In addition,qRT-PCR demonstrated over 7-fold increased expression of the ICAM-1 mRNAin HUVEC treated with Endothelin and BQ-788 compared to untreated HUVECor HUVEC in the presence of Endothelin alone, or Endothelin plus theETRA antagonist. Moreover, there was a decrease in the expression ofVE-Cadherin mRNA in BQ788-treated cells (FIG. 9). No specific changeswere detected in mRNA levels for ICAM-2, ICAM-3, E-selectin, JAM,CXCL-11, CCL-19, or CCL-21.

The ability of activated T cells to adhere to BQ-788-treated HUVEC wasalso tested. Human T cells activated with either PMA or CD3/CD28cross-linking exhibited increased adherence to HUVEC treated withEndothelin in the presence of BQ-788, compared to HUVEC treated withEndothelin alone, or treated with Endothelin and ETRA antagonist, oruntreated HUVEC (FIG. 9). T cell adherence to BQ788/Endothelin-treatedHUVEC was 40% as effective as TNF-alpha activation of HUVEC. Thus, underthe conditions utilized, BQ788 induces expression of ICAM-1 onendothelial cells and leads to increased T cell adhesion to tumorendothelium, playing a role in its increase of intratumoral T cells andenhancement of vaccine efficacy.

To further test the role of ETRB signaling in adhesion, the effects ofNO antagonist L-NAME and NO donor DETANO were tested under the aboveexperimental conditions. L-NAME restored T cell adhesion to HUVEC in thepresence of TNF- and ET-1, while DETANO mimicked the effects of ET-1.Thus, ET-1, through ETRB, downregulates the ability of endothelium torespond to inflammatory signals present in the tumor microenvironmentsuch as TNF-α, which is restored by blocking ETRB through BQ788.Further, an NO antagonist abrogated the effects of ET-1, while NO donorreproduced its effect, showing that NO plays a role in the inhibitoryeffect of ET-1. To further test whether ETRB signaling upregulates NO inendothelial cells, reactive oxidative species (ROS) were quantified inHUVEC. Exposure of HUVEC to rhET-1 upregulated ROS, while addition ofBQ788 abrogated such response to ET-1. Suppression of ROS by BQ788 wasas potent as bacterial LPS.

1. A method of treating or of enhancing an efficacy of an immunotherapyfor a solid tumor in a subject, comprising the step of contacting saidsubject with a compound or a composition that reduces an activity of orexpression of an Endothelin B receptor (ETRB).
 2. The method of claim 1,wherein said compound is BQ788; Bosentan (Tracleer™); tezosentan, or anantibody. 3.-12. (canceled)
 13. A method of treating or of enhancing anefficacy of an immunotherapy for a solid tumor in a subject, comprisingthe step of contacting said subject with a compound or composition thatreduces an activity of or expression of a protein selected from Musashi2, delta-like 1, Hairy/Enhancer of Split 1, MEG3, SEC61G, KIAA1609,ACTR6, clone LNG00414, ATP9A, IMAGE:23539, NCOA1, WIT1, PAPSS2, ALDOA,ZNF423, ENPP2, HSU79266, KIAA0146, IMAGE:1902075, EMX2, MYBL1,IMAGE:1660792, IMAGE:191524, IMAGE:2365035, TAF3, SLC1A4, and SGCB. 14.The method of claim 13, wherein said compound is an antibody. 15.-20.(canceled)
 21. A method of treating or of enhancing an efficacy of animmunotherapy for a solid tumor in a subject, comprising the step ofcontacting said subject with a compound or a composition that increasesan activity of or expression of a protein selected from a CASP8 andFADD-like apoptosis regulator (CFLAR) protein; estrogen receptor alpha(ESR1); caldesmin-1; adrenergic receptor B2 (ADRBK2); IMAGE:2755380,ZNFN1A5, LOC283663, IGLJ3, ZNF521, COL05405, CYP1B1, EIFSB,IMAGE:1518332, HSPC056, FLJ32949, IMAGE:244300, FLJ10330, C18orf14,IMAGE:2115041, GBP1, IMAGE:731714, SFRS1, NICAL, NOL7, MYCBP2,IMAGE:2275600, ADRBK2, EST366269, SCAP2, STK3, and AKAP10.
 22. Themethod of claim 21, wherein said compound is an antibody.
 23. (canceled)24. (canceled)
 25. A method of enhancing an efficacy of an immunotherapyfor a solid tumor in a subject, comprising the step of contacting saidsubject with a compound or a composition that reduces an expression oractivity of an endothelin-1 (ET-1) protein or that reduces interactionbetween an Endothelin B receptor (ETRB) and an endothelin-1 (ET-1),thereby enhancing an efficacy of an immunotherapy for a solid tumor in asubject.
 26. The method of claim 25, wherein said compound is anantisense compound or RNA inhibitory molecule or an antibody. 27.-31.(canceled)
 32. A method of enhancing an efficacy of an immunotherapy fora solid tumor in a subject, comprising the step of contacting saidsubject with a compound or a composition that increases an expression oran activity of an intercellular adhesion molecule 1 (ICAM-1 protein),thereby enhancing an efficacy of an immunotherapy for a solid tumor in asubject.
 33. The method of claim 32, wherein said compound is anantisense compound, RNA inhibitory molecule, or an antibody.